Treating multiple sclerosis

ABSTRACT

A method for treating multiple sclerosis comprises applying peripheral blood from a patient or subject to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a chemokine receptor, optionally the chemokine receptor CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 immobilized directly or indirectly on the support thus removing one or more chemokine receptor, optionally CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells from the peripheral blood of the patient or subject. Various companion diagnostic methods and useful binding reagents are also described.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.14/105,628, filed on Dec. 13, 2013, which is a continuation-in-part ofInternational Patent Application No. PCT/GB2012/051357, filed on Jun.13, 2012, which claims priority to U.S. Provisional Patent ApplicationNo. 61/496,442, filed on Jun. 13, 2011. U.S. patent application Ser. No.14/105,628 is also a continuation-in-part of International PatentApplication No. PCT/GB2012/051349, filed on Jun. 13, 2012, which claimspriority to U.S. Provisional Patent Application No. 61/496,167, filed onJun. 13, 2011. U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051348, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,288, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051351, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,242, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051350, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,209, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051355, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,195, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051345, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,228, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051352, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,264, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051346, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,184, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051353, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,329, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051356, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,377, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051354, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,352, filed on Jun. 13, 2011.The entire contents of each of these applications are fully incorporatedherein by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 21, 2017, isnamed P81602725US00-211226-9002-US01-SEQ-LIST-06-21-17.txt, and is27,709 bytes in size.

FIELD OF THE INVENTION

The various embodiments of the present invention relates to products forand methods of treating inflammatory conditions, such as multiplesclerosis, in particular active and stable relapsing-remitting multiplesclerosis, primary progressive, secondary progressive and progressiverelapsing multiple sclerosis. Companion diagnostics are also described.

BACKGROUND OF THE INVENTION

Multiple sclerosis is a neurodegenerative disease affecting the centralnervous system (CNS). Disease onset usually occurs in young adultsbetween the ages of 20 and 40, however, the precise underlying cause ofMS is unknown. There is currently no cure for this disabling disease andtreatment is primarily focussed on management of symptoms.

Irrespective of disease etiology, the immune system has been found toplay a central role in the pathogenesis of MS. In particular, thelesions that develop in the brain and/or spinal cord during diseaseprogression are frequently characterised by an excessive inflammatoryinfiltrate and the presence of autoreactive CD4+/CD8+ T cells andautoreactive B cells. Furthermore, ongoing assault of the CNS mediatedby a variety of inflammatory and/or immune cell types appears to be theprimary cause of the nerve damage and in particular, the axondemyelination, associated with this disease.

Apheresis is a treatment used for depletion of blood components, such asantibodies, low-density lipoproteins (LDL) and blood cells.Leukapheresis is the apheresis treatment used for removal of white bloodcells, leukocytes. The patient is connected to an extracorporeal bloodcirculating system; the blood is drawn from a vein in one arm, passedthrough a column device and returned into the other arm of the patient.WO2010/029317 describes apheresis columns useful for treatinginflammatory conditions including a chemokine immobilised on a solidsupport.

SUMMARY OF THE INVENTION

Chemokines are a class of cytokine molecules involved in cellrecruitment and activation in inflammation. Chemokines cause chemotaxisand activation of various subpopulations of cells in the immune system.The activity of chemokines is mediated primarily through tight bindingto their receptors on the surface of leukocytes. In certain embodimentsthe present invention is based on the realisation that the interactionbetween chemokines and cells expressing their receptors may be exploitedfor the treatment of multiple sclerosis. In particular, various types ofmultiple sclerosis, such as active and stable relapsing-remittingmultiple sclerosis include an inflammatory component. The inventors havedetermined that targeting increased recruitment of specific chemokinereceptor-expressing cells to the site of inflammation presents a newtherapeutic approach to treat such conditions. Moreover, in suchconditions, chemokine receptor expression on each cell may be increasedagain providing a therapeutic approach to treat such conditions. It isshown herein that subjects suffering from MS exhibit increased frequencyof chemokine receptor expressing cells in the peripheral blood, inparticular CCR2 and CCR6 expressing T lymphocytes, compared to healthycontrols. It is also shown herein that the CCR2 cells can be removedusing a suitable binding reagent, in particular MCP-1 (in biotinylatedform) immobilized on a suitable matrix. Similarly, it is shown hereinthat (the additional) CCR6-expressing cells can be depleted using asuitable binding reagent, in particular CCL20 (MIP-31), in biotinylatedform, immobilized on a suitable matrix.

Thus, in certain embodiments the invention serves to reduce therecruitment of inflammatory leukocytes, which express characteristicchemokine receptors, and possibly express characteristic chemokinereceptors at increased levels, to sites of inflammation linked tomultiple sclerosis such as active and stable relapsing-remittingmultiple sclerosis, primary progressive, secondary progressive andprogressive relapsing multiple sclerosis. This is achieved usingspecific binding reagents to capture specific chemokinereceptor-expressing inflammatory leukocytes from the patient.Accordingly, in certain embodiments the invention provides in a firstaspect a method for treating multiple sclerosis comprising applyingperipheral blood from a patient to an apheresis column loaded with asolid support comprising one or more binding reagents capable ofspecifically binding to one or more chemokine receptors, in particularto the chemokine receptors selected from CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9, immobilized directly or indirectly on the supportthus removing chemokine receptor, in particular one or more of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9, expressing cells from theperipheral blood of the patient. The peripheral blood from which thechemokine receptor expressing cells have been removed may then bereturned to the patient in order to complete the treatment. Theinvention may thus rely on a continuous extracorporeal circuit in someembodiments. Alternatively, in certain embodiments the invention maycomprise steps of obtaining peripheral blood from the patient, applyingthe peripheral blood to the column and subsequently returning theperipheral blood from which the chemokine receptor expressing cells havebeen removed to the patient.

As shown herein, suitable binding reagents can be immobilized onto asolid support, either directly or indirectly, to generate an apheresiscolumn suitable for capturing relevant chemokine receptor-expressingcells. Where increased levels of chemokine receptor expression areobserved, such cells may be preferably removed from the peripheral bloodusing the columns of the various embodiments of the invention. Thus, themethods of the various embodiments of the invention may preferablytarget one or more of CCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi),CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi) cells as defined herein forremoval from the peripheral blood. “High” expression may be determinedaccording to standard flow cytometry techniques. The level is measuredrelative to levels of expression of the chemokine receptor in cellstaken from a healthy subject. The attached FIG. 13 provides an exampleof a gating strategy.

In other embodiments the invention further provides a binding reagentcapable of specifically binding to one or more chemokine receptors, inparticular to a chemokine receptor selected from CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9, for use in the treatment of multiple sclerosis,wherein the one or more binding reagents is immobilized, directly orindirectly, on a solid support contained within an apheresis column, towhich is applied peripheral blood from a patient thus removing one ormore chemokine receptor/CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells from the peripheral blood of the patient. In certainembodiments the invention also provides for use of one or more bindingreagents capable of specifically binding to a chemokine receptor/thechemokine receptor CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 foruse in the manufacture of an apheresis column for treatment of multiplesclerosis, wherein the one or more binding reagents is immobilized on asolid support contained within the apheresis column, to which is appliedperipheral blood from a patient thus removing one or more chemokinereceptor/CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells from the peripheral blood of the patient.

All embodiments described in respect of the methods of treatment of thevarious embodiments of the invention apply to these aspects mutatismutandis and are not repeated for reasons of conciseness. Thus, thefollowing discussion made with reference to the methods of treatment isalso applicable to the medical use aspects of the various embodiments ofthe invention.

In certain embodiments the invention aims to treat multiple sclerosis.By treatment is meant a reduction in the specific chemokine receptorexpressing cells in the peripheral blood of the patient. The reductionmay comprise a reduction in cells that express chemokine receptors, inparticular one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9, at increased levels in diseased patients. The patient is typicallya human patient but the term patient may include both human andnon-human animal subjects in some embodiments. In the context of thevarious embodiments of the present invention, this typically involves areduction in one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 expressing cells, such as one or more of “CCR2^(hi), CCR6^(hi),CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)” expressingcells, in the peripheral blood of the patient. The CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cells comprise, consistessentially of or consist of monocytes, lymphocytes, neutrophils,macrophages, eosinophils and basophils, in certain embodiments. Inspecific embodiments the cells removed in order to treat MS comprise Tlymphocytes, in particular CCR2 and/or CCR6 expressing T lymphocytes.

Monocytes are produced by the bone marrow from haematopoietic stem cellprecursors called monoblasts. Monocytes may differentiate intomacrophages or dendritic cells. Monocytes and their macrophage anddendritic cell progeny serve a number of functions in the immune systemincluding phagocytosis, antigen presentation and cytokine production.Monocytes may be characterized with reference to expression of the cellsurface marker CD14, optionally together with CD16. Classical monocytesmay be characterized by high level expression of the CD14 cell surfacereceptor (CD14++ CD16− monocyte). Non-classical monocytes may becharacterized by low level expression of CD14 and with additionalco-expression of the CD16 receptor (CD14+CD16++ monocyte). Intermediatemonocytes may be characterized by high level expression of CD14 and lowlevel expression of CD16 (CD14++CD16+ monocytes). Macrophages arederived from monocytes and are responsible for protecting tissues fromforeign substances. They are cells that possess a large smooth nucleus,a large area of cytoplasm and internal vesicles for processing foreignmaterial. The term “macrophage” may refer to a monocyte-derived cellexpressing one or more of the following cell surface markers CD14,CD11b, Lysozyme M, MAC-1/MAC-3 and CD68. The term macrophage includesboth activated and un-activated macrophages. Activated macrophages maybe characterized by expression of one or more of CD69, ENG, FCER2 andIL2RA, HLA-DR, CD86. Un-activated macrophages have not yet receivedactivating signals through for example TLR receptors and therefore theyexpress less activation markers on the cell surface which correlateswith lesser maturation. M1 macrophages may be characterized byexpression of one or more of CD16⁺CD32⁺CD64⁺ and secrete mainly IL-23and IL-1, TNF, IL-6 and high levels of IL-12 and in addition effectormolecules such as iNOS and ROI. M1 macrophages have cytotoxic featuresas opposed to M2 macrophages. M2 macrophages may be characterized byexpression of one or more of SRA/B⁺CD163⁺MR⁺CD14⁺ and express TGFβ,IL-10 and IL-1Ra. Tumour associated macrophages (TAMs) share manycharacteristics with the M2 macrophages and are considered as M2polarised macrophages. The M1/M2 paradigm can also be found in monocytesubsets where CD14⁺CD16⁻CXC3R1^(low) monocytes are considered the“inflammatory” subset and the CD14^(low)CD16⁺CXC3R1^(high) are“resident” monocytes.

The three major types of lymphocyte are T cells, B cells and naturalkiller (NK) cells. The term “T-lymphocyte” includes CD4⁺ T cells such asT helper cells (Th1 cells and Th2 cells), and CD8⁺ T cells such ascytotoxic T cells. Th1 cells may be characterized by expression of CCR5and/or by production of IFN-γ. Th2 cells may be characterized byexpression of CCR3 and/or by production of IL-4.

The claimed methods may target eosinophils. The name “eosinophil”derives from the eosinophilic “acid-loving” properties of the cell.Normally transparent, it is this affinity that causes them to appearbrick-red after staining with eosin, a red dye, using the Romanowskymethod. The staining is concentrated in small granules within thecellular cytoplasm, which contain many chemical mediators, such ashistamines and proteins such as eosinophil peroxidase, ribonuclease(RNase), deoxyribonucleases, lipase, plasminogen, and major basicprotein. These mediators are released by a process called degranulationfollowing activation of the eosinophil, and are toxic to both parasiteand host tissues.

Eosinophils develop and mature in bone marrow. They differentiate frommyeloid precursor cells in response to the cytokines interleukin 3(IL-3), interleukin 5 (IL-5), and granulocyte macrophagecolony-stimulating factor (GM-CSF). Eosinophils produce and store manysecondary granule proteins prior to their exit from the bone marrow.After maturation, eosinophils circulate in blood and migrate toinflammatory sites in tissues in response to chemokines such as CCL11(eotaxin-1), CCL24 (eotaxin-2), CCL5 (RANTES) and MCP1/4. Eosinophilsmay be activated by Type 2 cytokines released from a specific subset ofhelper T cells (Th2); IL-5, GM-CSF, and IL-3 are important foreosinophil activation as well as maturation. CD44 and CD69 have beenshown to represent different types of cell-surface activation markersfor human eosinophils. CD69 is absent from “fresh” eosinophils butexpressed following activation (using cytokines). CD44 on the other handis constitutively expressed but expression is significantly up-regulatedin response to activation (Matsumoto et al., Am. J. Respir. Cell Mol.Biol., Volume 18, Number 6, June, 1998 860-866). Cell specific markersfor eosinophils include CD9 and CDw125.

Basophils may also be known as basophil granulocyte. In contrast toeosinophils, these leukocytes are basophilic, i.e., they are susceptibleto staining by basic dyes. Basophils contain large cytoplasmic granuleswhich obscure the cell nucleus under the microscope. However, whenunstained, the nucleus is visible and it usually has 2 lobes. Basophilsstore histamine, which is secreted by the cells upon stimulation.

Basophils have protein receptors on their cell surface that bind IgE, animmunoglobulin involved in macroparasite defense and allergy. It is thebound IgE antibody that confers a selective response of these cells toenvironmental substances, for example, pollen proteins or helminthantigens. Recent studies in mice suggest that basophils may alsoregulate the behavior of T cells and mediate the magnitude of thesecondary immune response. Basophils may display an immunophenotypebased upon expression (or lack thereof, indicated as “+” or “−”respectively of one or more of the following markers: FcεRI+, CD123,CD49b(DX-5)+, CD69+, Thy-1.2+, 2B4+, CD11bdull, CD117(c-kit)−, CD24−,CD19−, CD80−, CD14−, CD23−, Ly49c−, CD122−, CD11c−, Gr-1−, NK1.1−,B220−, CD3−, γδTCR−, αβTCR−, α4 and β4-integrin negative.

When activated, basophils degranulate to release histamine,proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes(e.g. elastase and lysophospholipase). They also secrete lipid mediatorslike leukotrienes, and several cytokines. Histamine and proteoglycansare pre-stored in the cell's granules while the other secretedsubstances are newly generated. Each of these substances contributes toinflammation. Recent evidence suggests that basophils are an importantsource of the cytokine, interleukin-4, perhaps more important than Tcells. Interleukin-4 is considered one of the critical cytokines in thedevelopment of allergies and the production of IgE antibody by theimmune system. There are other substances that can activate basophils tosecrete which suggests that these cells have other roles ininflammation.

The various embodiments of the methods of the invention may involvespecific binding interactions with any one or more of these furthercell-surface (and cell-specific) markers in addition to the removalbased upon binding to CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9.Suitable binding reagents can be prepared to specifically bind to thesecell-surface markers. The discussion of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 specific binding reagents thus applies mutatismutandis.

CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressed on theseaforementioned cells are bound by chemokines such as monocytechemoattractant protein-1 (MCP-1), MCP-2, MCP-3, MCP-4, MCP-5,MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC) and RANTES.Eotaxin (aka CCL11 binds CCR3 only), eotaxin-2 (aka CCL24 binds CCR3only), eotaxin-3 (aka CCL26 binds CCR3 only), RANTES (CCL5 ispromiscuous CCR1, CCR3, CCR5), MCP-2, (aka CCL8 is promiscuous) MCP-3(aka CCL7 is promiscuous), MCP-4 (aka CCL13 is promiscuous), MIP-1a (akaCCL3 promiscuous CCR1, CCR3, CCR5), MEC (CCL28 binds CCR3 and CCR10),HCC-2 (CCL15 binds CCR1 and CCR3). Chemokines MIP1γ (CCL9), MRP-2(CCL10), MIp-1δ (CCL15) and CCL23 appear to bind CCR1 only, ChemokinesEotaxin, Eotaxin-2 only bind CCR3, Chemokine MIP1β (CCL4) only bindsCCR5. MCP-5 binds CCR2 only.

MCP-1 (CCL2) is a major chemoattractant for monocytes and memory T cellsby means of their binding to its specific cell-surface receptor,CC-chemokine receptor-2 (CCR2). CCR2 is the gene symbol approved by theHUGO Gene Nomenclature Committee for chemokine (C—C motif) receptor 2.The HGNC ID for this gene is 1603. The gene is located at chromosomeposition 3p21. The previous symbol and name for the gene is CMKBR2.Synonyms for this gene include CC-CKR-2, CD192, CKR2, FLJ78302 andMCP-1-R. The NCBI Reference Sequence is NM_001123041.2 as available on13 Jun. 2011, which is incorporated herein by reference in its entirety.

CCR1 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 1. The HGNC ID for this gene is 1602.The gene is located at chromosome position 3p21. The previous symbol andname CMKBR1, SCYAR1. Synonyms for this gene include CD191, CKR-1,MIP1aR. The Entrez Gene reference sequence for CCR1 is 1230 as availableon 13 Jun. 2011, which is incorporated herein by reference in itsentirety.

CCR3 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 3. The HGNC ID for this gene is 1604.The gene is located at chromosome position 3p21.3. The previous symboland name for the gene is CMKBR3. Synonyms for this gene includeCC-CKR-3, CD193 and CKR3. The Genbank reference sequence for CCR3 isAF247361.1 as available on 13 Jun. 2011, which is incorporated herein byreference in its entirety.

CCR5 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 5. The HGNC ID for this gene is 1605.The gene is located at chromosome position 3p21. The previous symbol andname for the gene is CMKBR5. Synonyms for this gene include CC-CKR-5,CD195 CKR-5, IDDM22 and CKR5. The Entrez Gene reference sequence forCCR5 is 1234 as available on 13 Jun. 2011, which is incorporated hereinby reference in its entirety.

CCR6 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 6. The HGNC ID for this gene is 1607.The gene is located at chromosome position 6q27. The previous symbol andname for the gene is STRL22. Synonyms for this gene include BN-1, CD196,CKR-L3, CMKBR6, DCR2, DRY-6, GPR-CY4, GPR29. The Genbank referencesequence for CCR6 is U68030.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCR9 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 9. The HGNC ID for this gene is 1610.The gene is located at chromosome position 3p22. The previous symbol andname for the gene is GPR28. Synonyms for this gene include CDw199,GPR-9-6. The Genbank reference sequence for CCR9 is AJ132337.1 asavailable on 13 Jun. 2011, which is incorporated herein by reference inits entirety.

CXCR3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) receptor 3. The HGNC ID for thisgene is 4540. The gene is located at chromosome position Xq13. Theprevious symbol and name for the gene is “G protein-coupled receptor 9”,GPR9. Synonyms for this gene include CD183, CKR-L2, CMKAR3, IP10-R andMigR. The Genbank reference sequence for CXCR3 is U32674.1 as availableon 13 Jun. 2011, which is incorporated herein by reference in itsentirety.

Treatment according to the various embodiments of the invention mayresult in alleviation or amelioration of symptoms, prevention ofprogression, regression of the condition, or complete recovery.Measurable parameters of successful treatment include one or more, up toall, of Multiple Sclerosis Severity Score or MRI. In specificembodiments, a single treatment is sufficient to cause a depletion ofaround 10%, 20%, 30%, 40%, 50%, 60% or 70%, or higher up to 80%, 90%,95% or more, or any range of values between and including these amounts,of one or more of a specific chemokine receptor, in particular one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9, expressingcells from the peripheral blood of the patient. In specific embodiments,at least around 50% depletion is achieved in a single treatment. Thus,successful treatment may be defined with reference to depletion of oneor more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells. Treatment may lead to depletion of between approximately 100 and500 million CCR2, CCR6, CCR3, CCR5, CCR1 and/or CCR9 expressing cells,such as monocytes, in certain embodiments and more particularly to about100, 150, 200, 250, 300, 350, 400, 450, or 500 million CCR2, CCR6, CCR3,CCR5, CCR1 and/or CCR9 expressing cells.

By binding to the column through the binding reagent-chemokine receptorinteraction, chemokine receptor expressing cells are immobilized. Theseimmobilized cells express further unoccupied chemokine receptors, whichmay be of the same or different type to those used for capture. Theseadditional chemokine receptors may permit circulating chemokines whichcontribute to the inflammatory condition to be captured from theperipheral blood. Thus, a reduction in circulating (specific) chemokinelevels may provide a measure of successful treatment.

The duration of treatment may be readily determined by one skilled inthe art and will depend upon factors such as the flow rate of theperipheral blood. Duration of treatment may be tied into monitoring ofthe treatment itself, with the treatment considered complete once ameasurable parameter of treatment has reached a defined threshold. Anysuitable parameter may be employed as discussed herein. Thus, forexample, treatment may be considered complete when a reduction in one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells, such as a 50% reduction in one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expressing cells, has been achieved. Theapheresis system may be operated at a flow rate of around 10-80 mL/min,or more specifically between around 20-70 mL/min, or between around30-60 mL/min. In specific embodiments, the treatment is performed for aperiod of around 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 etc.,or any range of values between and including these amounts, minutes. Thetreatment is typically not aimed to remove all of the cells expressingthe chemokine receptor in the peripheral blood, as a basal level ofthose cells is required in healthy subjects. However, it has been foundthat only low blood volumes need to be applied to the columns of thevarious embodiments of the invention in order to achieve effectivelevels of depletion of the chemokine receptor-expressing cells. Thus, incertain embodiments, around 10-80% or more specifically around 20, 30,40 or 50%, or any range of values between and including these amounts,of the patient's blood is applied to the column in a single treatment.The volume of blood circulated through the apheresis column or systemmay be in the region of around 1000-3000 ml, such as around 1000, 1200,1400, 1600, 1800 or 2000 ml or any range of values between and includingthese amounts. The treatment may be considered complete once this volumeof blood has been circulated. The patient may be administeredanticoagulants prior to each treatment session. A suitable solution,such as a sterile saline solution, optionally including an anticoagulantsuch as Heparin, may be used for priming the apheresis (extracorporeal)system. An additional volume of anticoagulant may be added to thecircuit at the start of each treatment session, for example as a bolusinjection.

In certain embodiments the invention relies upon a binding reagent whichis capable of specifically binding to a chemokine receptor. Thisspecific binding reaction permits cells expressing the chemokinereceptor to be removed from the peripheral blood of the patient when theblood is passed over the solid support upon or within which the bindingreagent is immobilized. Specific chemokine receptors of interest includeCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9. The binding reagent canbe any binding reagent capable of specifically binding to the receptorin question. By “specific binding” is meant that the binding reagentdisplays sufficient specificity of binding and appropriate bindingaffinity/kinetics to permit removal of cells expressing one or more ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 from the peripheralblood. Whilst it is not precluded that the binding reagent is capable ofbinding to other molecules, such as other chemokine receptors, thebinding reagent will preferentially bind to cells expressing one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 and in particular tocells expressing increased levels of one or more of CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 (as defined further herein). The bindingreagent capable of specifically binding to CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 may be either an agonist or an antagonist of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9. As the disease conditionrelies upon up-regulation of expression of or signaling via CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9, in certain embodiments the bindingreagent capable of specifically binding to CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 is an antagonist of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9., respectively. Chemokines are typically, although notnecessarily exclusively (particularly in the case of truncated ormodified forms) agonists of their cognate receptor and serve to activatethe cells expressing the relevant receptor, as would be appreciated byone skilled in the art. Antibodies against the relevant chemokinereceptor are generally considered to be antagonists, as would beappreciated by one skilled in the art. Specific examples of bindingreagents include proteins or polypeptides, such as antibodies andreceptor ligands, in particular chemokines. The binding reagent may be anucleic acid molecule in certain embodiments. In some embodiments, thenucleic acid is an aptamer. Nucleic acid aptamers are polynucleotides ofapproximately 15-40 nucleotides long. Nucleic acid aptamers can be madeusing the SELEX process (systemic evolution of ligands by exponentialenrichment) or any other process known to those of skill in the art.

In other embodiments, the binding reagent may be a peptide, and incertain instances, a peptide aptamer. Peptide aptamers are artificialrecognition molecules that consist of a variable peptide sequenceinserted into a constant scaffold protein (Baines I C, Colas P. Peptideaptamers as guides for small molecule drug discovery. Drug Discov Today.2006; 11:334-341, incorporated herein by reference). A number ofmethodologies, such as phage display, ribosome display and yeasttwo-hybrid screening systems are available for screening a library ofpotential peptide-based binding agents. Similarly protein scaffoldsbased on domains such as fibronectins, ankyrin repeats, protein A, SH3domains, lipocalins and ubiquitin can be used as the binding agent.Again a number of technologies such as phage display and ribosomedisplay are available for screening a library of protein—based bindingagents. Similarly, libraries of candidate chemical compounds can bescreened for specific binding to the relevant chemokine receptor usingsuitable screening techniques known in the art, which may be highthroughput screens in certain embodiments. The candidate binding agentmay be immobilized on a solid support and the ability of the agent tospecifically retain cells expressing the chemokine receptor of interestor labelled chemokine receptors determined. A range of cell types may beapplied to the solid supports to confirm specificity of binding, oralternatively a mixed sample (such as peripheral blood) may be appliedto the solid support. Retention of the cell type of interest (expressingthe appropriate chemokine receptor) can be confirmed to identifysuitable binding agents. A range of small-molecule antagonists of CCR-2are discussed by Xia M and Sui Z in Expert Opin Ther Pat. 2009 March;19(3):295-303—Recent developments in CCR2 antagonists, and incorporatedherein by reference.

In the context of the various embodiments of the present invention theterm “chemokine” also comprises biotinylated or otherwise labelledchemokines. The term “chemokine” also comprises modified and truncatedversions of the chemokine and chemokine fragments with the proviso thatthe modified or truncated form retains its ability to bind to itscognate receptor (and thus remains functional in the context of variousembodiments of the the invention). The chemokine does not necessarilyneed to retain biological activity as it is specific binding affinityfor CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 that is required. Incertain embodiments, the chemokine lacks biological activity, forexample in terms of activation of the (CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9) receptor. Modifications may be made to improveprotein synthesis, for example uniformity of product and yield. As knownto those skilled in the art, exemplary modifications may comprise aminoacid additions, substitutions, deletions or other modifications to oneor more amino acids in the chemokine. Modifications may comprisesubstitution of the wild type amino acid with non-natural amino acidssuch as norleucine (NLeu) and derivatized amino acids such aspyroglutamic acid (pyroGlu). Such modifications may be made to minimizeside-product formation during storage and use of the columns of thevarious embodiments of the invention. Modifications may be made toimprove labelling, for example inclusion of a polyethylene glycol (PEG)spacer to facilitate biotinylation. The biotinylation and/or conjugationwith fluorochromes or other labelling groups of the chemokine isperformed in a manner which does not substantially affect the receptorbinding capacity. Site specific biotinylation or other labelling ispreferred as non-selective labelling of chemokines may compromisereceptor binding activity. Bioinylation or other labelling is generallypreferred at or towards the C-terminus of the protein as the inventorshave found that modifications in this area are generally well tolerated(in terms of a minimal effect on receptor binding capability).Biotinylation may be carried out site-specifically at any suitable aminoacid. Examples of suitable amino acids include lysine, diaminopropionicacid and ornithine. Generally, reference may be made to Natarajan S etal, Int. J. Pept. Protein Res., 1992, 40, 567-74; Baumeister B, Int. J.Peptide Res. And Therapeutics, 2005, 11, 139-141; Bioconjugatetechniques 2^(nd) edition, Greg T. Hermanson, incorporated by referenceherein in its entirety.

Truncations may involve deletion of either N or C terminal amino acidsas appropriate, or both. Typically, the truncated version will retainthe residues required for the chemokine to fold correctly, for exampleto retain a chemokine fold structure, consistent with the requirementthat a truncated version must retain the ability to bind to the relevantreceptor (expressed by (on the surface of) a leukocyte). Chemokinemolecules typically include disulphide bonds between the 1^(st) and3^(rd) and 2^(nd) and 4^(th) cysteine residues respectively, as would beunderstood by one skilled in the art. Where sequences are presentedherein, it is assumed that these disulphide bonds will form in thefolded protein (unless otherwise stated). Truncated versions maycomprise anywhere between 1 and 100 less amino acids, such as 1, 2, 3,4, 5 etc amino acids, than the wild type amino acid sequence in certainembodiments. Of course, truncated versions may comprise furthermodification as detailed herein. The modified or truncated version mayhave 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moreoverall amino acid sequence identity with the full length wild typechemokine (where a deletion is counted as a difference in amino acidsequence) in certain embodiments. Over the common sequence between themolecules (i.e the amino acids that have not been deleted), there may be80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acidsequence identity in certain embodiments. Sequence identity may bedetermined using known algorithms, such as BLAST or GAP analysis (GCGProgram) (applying default settings), which are freely available.Chemokines may lack the N-terminal signal peptide which is cleaved offduring synthesis in vivo.

Specific chemokines useful in the various embodiments of the presentinvention include MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG(CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES. Both MCP-1 andMCP-5 bind solely to the chemokine receptor CCR2 and so these chemokinesmay be preferred in some embodiments. Each chemokine is able to bind toa chemokine receptor implicated in multiple sclerosis.

The modified and truncated chemokines described in greater detail herein(with reference to the relevant amino acid sequences, as set forth inthe SEQ ID NOs and accompanying experimental examples) may each beapplied according to the present invention. Such modified forms mayinstruct the skilled person regarding additional modified forms of thesame and other chemokines which may be suitable for use in theinvention. Chemokines show variable sequence homology varying from lessthan 20% to over 90% but all share very similar tertiary structuresconsisting of a disordered N-terminus, followed by a long loop (theN-loop) that ends in a 3₁₀ helix, a 3-stranded β-sheet and a C-terminalhelix. The overall topology is stabilsed by disulphide bonds. Thiscommon tertiary structure is a common feature of the chemokine proteinfamily (Fernandez E J and Lolis E., Annu. Rev. Pharmacol. Toxicol., 202,42, 469-99; Allen S J et al, Annu. Rev. Immunol., 2007, 25, 787-820,incorporated herein by reference).

Truncations within this N-terminal region can maintain binding to thereceptor but can lead to a change or loss of function (for examplesZhang Y J et al, J. Biol. Chem., 1994, 269, 15918; Gong J-H andClark-Lewis I., J. Exp. Med., 1995, 181, 631-640; Fernandez E J andLolis E., Annu. Rev. Pharmacol. Toxicol., 202, 42, 469-99; Allen S J etal, Annu. Rev. Immunol., 2007, 25, 787-820, each of which isincorporated herein by reference).

Truncations at the C-terminus of the chemokine can also be made andmaintain receptor binding activity (Treating Inflammatory Disorders, OlaWinqvist and Graham Cotton, WO2010/029317, incorporated herein byreference in its entirety).

In other embodiments, fragments and variants of chemokines are used inthe devices and methods as disclosed herein. More particularly, suchfragments and variants retain the ability to specifically bind to theircognate chemokine receptor. Chemokines are known by those skilled in theart to share specific receptor binding domains, including a similarmonomeric fold, characterized, for example, by a disorderedamino-terminal domain, followed by a conserved core region, consistingof the so called “N-loop,” three anti-parallel β-strands, and acarboxyl-terminal α-helix. While not being bound by theory, it isbelieved that the chemokine-chemokine receptor interaction is a two-stepmechanism, in which the core of the chemokine interacts first with abinding site formed by the extracellular domains of the receptor, whileanother interaction is formed between the chemokine N terminus and asecond binding site on the receptor in order to trigger receptoractivation. Thus, a “fragment,” such as a functional fragment of achemokine is intended to mean a portion of the amino acid sequence ofthe protein that retains binding for its cognate receptor. The fragmentmay include, for example, the monomeric fold region, or portions thereofsuch as the amino-terminal domain, the conserved core region and/or the“N-loop,” the anti-parallel β-strands, and/or the carboxyl-terminalα-helix or combinations and portions thereof.

Further, it is recognized that a polypeptide can be considerably mutatedwithout materially altering one or more of the polypeptide's functions,for example, without altering specific binding and/or the folding of theprotein. The genetic code is well known to be degenerate, and thusdifferent codons encode the same amino acids. Even where an amino acidsubstitution is introduced, the mutation can be conservative and have nomaterial impact on the essential functions of a protein (see forexample, Stryer, Biochemistry 4th Ed., W. Freeman & Co., New York, N.Y.,1995). This includes, for example, the ability of the protein to bindand interact with other proteins, such as a truncated chemokine bindingto its cognate receptor.

In some examples, part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. For example, the deletionof between about 1 and about 20 amino acids on the C- and/or N-terminus,such as deletions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids at the C- and/or N-terminus,can result in a chemokine that retains function, such as specificbinding of its cognate receptor. Such truncations can retain the fullfunction of an entire protein, and/or can allow for retained functionssuch as protein-protein interactions as in the case of ligand-receptorinteractions. Chemokines having deletions of a small number of aminoacids, for example, less than about 20% (such as less than about 18%,less than about 15%, less than about 10%, less than about 8%, less thanabout 5%, less than about 2%, or less than about 1%) of the total numberof amino acids in the wild type chemokine can also be used in themethods and devices disclosed herein. Moreover, insertions or additionscan be made in the polypeptide chain for example, adding epitope tags,without impairing or eliminating its functions (Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publ. Assoc. andWiley-Intersciences, 1998). Other modifications that can be made withoutmaterially impairing one or more functions of a polypeptide include, forexample, in vivo or in vitro chemical and biochemical modifications orthe incorporation of unusual amino acids. In some examples, a functionalfragment of a chemokine may consist of about 10 or more, about 25 ormore, about 50 or more, about 75 or more, about 100 or more, about 125or more, about 150, about 175 or more, or about more or 200 or moreamino acid residues of a chemokine amino acid sequence.

In some examples, the chemokine or a functional fragment thereof has anamino acid that has at least about 60% or 65% sequence identity, about70% or 75% sequence identity, about 80% or 85% sequence identity, about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identityover its full length as compared to a reference sequence, such as thosedetailed herein, for example using the NCBI Blast 2.0 gapped BLAST setto default parameters. Alignment may also be performed manually byinspection. One or more conservative amino acid modifications can alsobe made in the chemokine amino acid sequence, whether an addition,deletion or modification, that does not substantially alter the3-dimensional structure of the polypeptide or its ability to bind to thecognate receptor. For example, a conservative amino acid substitutiondoes not affect the ability of the chemokine to specifically bind itscognate receptor. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Peptides, such as chemokines and fragments thereof, can be modified by avariety of chemical techniques to produce derivatives having essentiallythe same activity or function-such as binding to a cognate receptor—asthe unmodified peptides, and optionally having other desirableproperties. For example, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, may be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C1-C16ester, or converted to an amide of formula NR1R2 wherein R1 and R2 areeach independently H or C1-C16 alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C1-C16 alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C1-C16alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, C1-C16alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C2-C4 alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides of this disclosure toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a C- or N-terminal cysteinecan be added to the peptide, so that when oxidized the peptide willcontain a disulfide bond, generating a cyclic peptide. Other peptidecyclizing methods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present disclosure, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid side chains, resulting in such peptido- and organomimetics ofthe proteins of this disclosure. For computer modeling applications, apharmacophore is an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido- andorganomimetics can be designed to fit each pharmacophore with currentcomputer modeling software (using computer assisted drug design orCADD). See Walters, “Computer-Assisted Modeling of Drugs”, in Klegerman& Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press:Buffalo Grove, Ill., pp. 165 174 and Principles of Pharmacology Munson(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Alsoincluded within the scope of the disclosure are mimetics prepared usingsuch techniques.

Amino acids in a peptide, polypeptide, or protein generally arechemically bound together via amide linkages (CONH). Additionally, aminoacids may be bound together by other chemical bonds. For example,linkages for amino acids or amino acid analogs can include CH2NH—,—CH2S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and—CHH2SO— (These and others can be found in Spatola, in Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J PeptProt Res 14:177-185, 1979; Spatola et al. Life Sci 38:1243-1249, 1986;Harm J. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med.Chem. 23:1392-1398, 1980; Jennings-White et al. Tetrahedron Lett23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; andHruby Life Sci 31:189-199, 1982.

Chemokines MIP1γ (CCL9), MRP-2 (CCL10), MIp-1δ (CCL15) and CCL23 appearto bind CCR1 only, Chemokines Eotaxin, Eotaxin-2 only bind CCR3,Chemokine MIP1β (CCL4) only binds CCR5, Chemokine MIP3a (CCL20) onlybinds to CCR6, More specifically, each of MCP-1, MCP-2, MCP-3, MCP-4,MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC) and RANTESare useful for removing one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 expressing cells from the blood of a patient. Inspecific embodiments, the chemokine is selected from MCP-1, MCP-2,MCP-3, MCP-4 and MCP-5 and the chemokine receptor is CCR2. In otherembodiments, the chemokine is MIP-3a and the chemokine receptor is CCR6.In still further embodiments, the chemokine is RANTES and the chemokinereceptor is selected from CCR3, CCR1, CCR5 or CCR9.

The chemokines described in greater detail herein (with reference to therelevant figures and amino acid sequences, as set forth in the SEQ IDNOs and the corresponding experimental examples) may each be appliedaccording to the present invention.

CCL2 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 2, also known as MCP-1. The HGNC ID forthis gene is 10618. The gene is located at chromosome position17q11.2-q21.1. The previous symbol and name for the gene is SCYA2 “smallinducible cytokine A2 (monocyte chemotatic protein 1, homologus to mouseSig-je)”. Synonyms for this gene include GDCF-2, HCl 1, MCP1, MGC9434,SMC-CF, “monocyte chemoattractant protein-1”, “monocyte chemotactic andactivating factor”, “monocyte chemotactic protein 1, homologous to mouseSig-je”, “monocyte secretory protein JE”, “small inducible cytokinesubfamily A (Cys-Cys), member 2”. The Genbank reference sequence forCCL2 is BC009716.1 as available on 13 Jun. 2011, which is incorporatedherein by reference in its entirety.

CCL8 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 8, also known as MCP-2. The HGNC ID forthis gene is 10635. The gene is located at chromosome position 17q11.2.The previous symbol and name for the gene is SCYA8, “small induciblecytokine subfamily A (Cys-Cys), member 8 (monocyte chemotactic protein2)”. Another synonym for this gene is HC14. The Genbank referencesequence for CCL8 is X99886.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL7 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 7, also known as MCP-3. The HGNC ID forthis gene is 10634. The gene is located at chromosome position17q11.2-q12. The previous symbol and name for the gene is SCYA6, SCYA7,“small inducible cytokine A7 (monocyte chemotactic protein 3)”. Synonymsfor this gene include FIC, MARC, MCP-3, MCP3, NC28, “monocytechemoattractant protein 3”, “monocyte chemotactic protein 3”. TheGenbank reference sequence for CCL7 is AF043338 as available on 13 Jun.2011, which is incorporated herein by reference in its entirety.

CCL13 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 13, also known as MCP-4. TheHGNC ID for this gene is 10634. The gene is located at chromosomeposition 17q11.2-q12. The previous symbol and name for the gene isSCYA6, SCYA7, “small inducible cytokine A7 (monocyte chemotactic protein3)”. Synonyms for this gene include FIC, MARC, MCP-3, MCP3, NC28,“monocyte chemoattractant protein 3”, “monocyte chemotactic protein 3”.The Genbank reference sequence for CCL13 is AJ001634 as available on 13Jun. 2011, which is incorporated herein by reference in its entirety.

MCP-5 is a mouse chemokine in the CC chemokine family. It is also knownas Chemokine (C—C motif) ligand 12 (CCL12) and, due to its similaritywith the human chemokine MCP-1, sometimes it is called MCP-1-relatedchemokine. The gene for MCP-5 is found in a cluster of CC chemokines onmouse chromosome 11. The NCBI reference sequence for CCL12 isNC_000077.5 as available on 13 Jun. 2011, which is incorporated hereinby reference in its entirety.

CCL20 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 20, also known as MIP-3alpha.The HGNC ID for this gene is 10619. The gene is located at chromosomeposition 2q33-q37. The previous symbol and name for the gene is SCYA20,“small inducible cytokine subfamily A (Cys-Cys), member 20”. Synonymsfor this gene include CKb4, exodus-1, LARC, MIP-3a, ST38. The Genbankreference sequence for CCL20 is D86955.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CCL5 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 5, also known as RANTES. The HGNC IDfor this gene is 10632. The gene is located at chromosome position17q11.2-q12. The previous symbol and name for the gene is D17S136E,SCYA5, “small inducible cytokine A5 (RANTES)”.

Synonyms for this gene include “beta-chemokine RANTES”, MGC17164,RANTES, “regulated upon activation, normally T-expressed, and presumablysecreted”, “SIS-delta”, SISd, “small inducible cytokine subfamily A(Cys-Cys), member 5”, “T-cell specific protein p288”, “T-cell specificRANTES protein”, TCP228. The Genbank reference sequence for CCL5 isAF043341.1 as available on 13 Jun. 2011, which is incorporated herein byreference in its entirety.

CXCL11 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 11. The HGNC ID for thisgene is 10638. The gene is located at chromosome position 4q21. Theprevious symbol and name for the gene is SCYB9B, SCYB11, “smallinducible cytokine subfamily B (Cys-X-Cys), member 11”. Synonyms forthis gene include b-R1, H174, I-TAC, IP-9. The Genbank referencesequence for CXCL11 is U66096.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL25 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 25. The HGNC ID for this geneis 10624. The gene is located at chromosome position 19p13.2. Theprevious symbol and name for the gene is SCYA25, “small induciblecytokine subfamily A (Cys-Cys), member 25”. Synonyms for this geneinclude “Ck beta-15”, Ckb15, TECK, “TECKvar”, “thymus expressedchemokine”. The Genbank reference sequence for CCL25 is U86358.1 asavailable on 13 Jun. 2011, which is incorporated herein by reference inits entirety.

CXCL10 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 10. The HGNC ID for thisgene is 10637. The gene is located at chromosome position 4q21. Theprevious symbol and name for the gene is INP10, SCYB10, “small induciblecytokine subfamily B (Cys-X-Cys), member 10”. Synonyms for this geneinclude C7, crg-2, gIP-10, IFI10, IP-10, mob-1. The Genbank referencesequence for CXCL10 is X02530.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

Examples of suitable modified chemokines of the various embodiments ofthe invention containing modifications and/or truncations andspecifically adapted for use in the invention are described in detailherein. MCP-1 has been produced with residue 75, which may be a lysine(or any other amino acid such as ornithine and diaminopropionic acid,that may be biotinylated), as the site of biotinylation on the chemokine(numbering based upon the mature protein having the amino acid sequenceof SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on asolid support (via a biotin-avidin interaction). The basic amino acidsequence of MCP-1, including a 23 amino acid leader sequence is setforth as SEQ ID NO: 1. The amino acid sequence of the mature protein isset forth as SEQ ID NO: 2. The inventors have determined that chemokinesmay display improved binding properties where the chemokine isbiotinylated via a spacer group. The spacer may prevent the biotin groupfrom impacting on the binding affinity of the chemokine. Any suitablespacer group may be employed. Further modifications may provide themolecule with advantageous properties. The invention also relates toderivatives of truncated MCP-1 chemokines. The amino acid sequence ofthe truncated version is set forth as SED ID NO:3.

Accordingly, in certain embodiments the invention also provides amodified MCP-1 chemokine comprising, consisting essentially of orconsisting of the amino acid sequence set forth as SEQ ID NO: 1, SEQ IDNO: 2 or SEQ ID NO: 3 in which one or more of the followingmodifications have been made:

a) the glutamine residue 1 of SEQ ID NO: 2 has been replaced withpyroglutamineb) the C terminus is produced as an amide derivative (this may beachieved by synthesis on an amide linker)c) the (C terminal region) residue at position 98 of SEQ ID NO: 1 orposition 75 of SEQ ID NO:2 or position 67 of SEQ ID NO: 3, which may bea lysine or ornithine residue, is biotinylated, optionally via a spacergroup, in order to permit immobilization of the chemokine on a solidsupport; and/ord) the methionine residue at position 87 of SEQ ID NO: 1 or position 64of SEQ ID NO: 2 or position 56 of SEQ ID NO: 3 has been replaced withnorleucine.

The (C terminal region) amino acid, which may be a lysine residue or afunctional equivalent, at position 98 of SEQ ID NO: 1 or position 75 ofSEQ ID NO:2 or position 67 of SEQ ID NO: 3 may be biotinylated via asuitable spacer group, such as a polyethylene glycol (PEG) spacer group,in order to permit immobilization of the chemokine on a solid support.In specific embodiments, the PEG spacer is 3,6-dioxo aminooctanoic acid.The sequence and biotinylation of the modified MCP-1 chemokines of theinvention are shown in FIGS. 7 to 9 respectively. The modified MCP-1chemokines may be agonists or antagonists of CCR2 activity. They can betested for activity in a suitable assay, such as cell-based assays. Inparticular, agonist and antagonist properties may be determined infunctional cell-based assay on human CCR2 receptor.

MCP-5 only binds CCR2 and should be selective in its removal of CCR2expressing cells. The full length amino acid sequence, including thesignal peptide, is set forth as SEQ ID NO: 4. The amino acid sequence ofN-terminal processed MCP-5 chemokine is 82 amino acids long and is setforth as SEQ ID NO: 5. An amino acid sequence alignment suggests thatMCP-5 has a C-terminal extension when compared to the amino acidsequence of MCP-1. The results of this alignment are shown in FIG. 10.C-terminal truncated versions of MCP-5 can thus be synthesised. Thistruncated version will comprise, consist essentially of or consist ofMCP-5 residues 1-76, set forth as SEQ ID NO: 6.

Accordingly, in certain embodiments the invention also provides amodified MCP-5 chemokine comprising the amino acid sequence set forth asSEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in which the isoleucineresidue at position 97 of SEQ ID NO: 4 or at position 75 of SEQ ID NO: 5or SEQ ID NO: 6 has been replaced with lysine (or a functionalequivalent such as ornithine or diaminopropionic acid, which can bebiotinylated). In certain embodiments, the modified MCP-5 chemokinecomprises, consists essentially of or consists of the amino acidsequence of SEQ ID NO: 7. The modified MCP-5 chemokine may bebiotinylated at the lysine (or a functional equivalent) residue atposition 97 of SEQ ID NO: 4 or at position 75 of SEQ ID NO: 5 or SEQ IDNO: 6. Biotinylation may be via a suitable spacer group. Specificexamples of the spacer group include a PEG spacer, optionally 3,6-dioxoaminooctanoic acid. In some embodiments, the C terminus is produced asan amide derivative. This may be achieved by synthesis on an amidelinker. In certain embodiments, the modified MCP-5 chemokine comprises,consists essentially of or consists of the sequence and biotinylationshown in FIG. 11. The modified MCP-5 chemokine may be an agonist or anantagonist of CCR2 activity. They can be tested for activity in asuitable assay, such as cell-based assays. In particular, agonist andantagonist properties may be determined in a functional cell-based assayon human CCR2 receptor.

An example of a CCL25 chemokine of the various embodiments of theinvention containing both modifications and a truncation andspecifically adapted for use in the invention is described inWO2010/029317, incorporated herein by reference. Reference may also bemade to Example 12 below and SEQ ID NOs 23 to 25.

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 8 below). Themodified CCL2 (MCP-1) corresponds to residues 1 to 76 of the full lengthmature protein (and lacks the N-terminal signal peptide of 23 aminoacids, which is cleaved off) and thus retains the chemokine fold. TheGln at the N-terminus of the protein (Gln1) is substituted withpyroglutamine to prevent mixed species of N-terminal Gln and pyroGlubeing generated (SEQ ID NO: 8). This improves the yield of synthesis andensures a homogeneous chemokine preparation through column manufactureand use. FmocLys(ivDde)-OH is incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 9).A suitable spacer, such as a PEG spacer, may be incorporated between theε-amino functionality and the biotin. Thus, the invention relates to amodified chemokine, including a biotinylated version, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 10:

H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT-NH₂

X1=pyroGlu (but may remain as Gln in some embodiments)

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 3 below). Themodified CCL8 (MCP-2) corresponds to residues 1 to 76 of the full lengthmature protein (and lacks the N-terminal signal peptide of 23 aminoacids, which is cleaved off) and thus retains the chemokine fold. TheGln at the N-terminus of the protein is subject to pyroGlu formationunder physiological conditions. Thus Gln1 of the sequence is substitutedwith pyroglutamine to prevent mixed species of N-terminal Gln andpyroGlu being generated (SEQ ID NO: 11). This improves the yield ofsynthesis and ensures a homogeneous chemokine preparation through columnmanufacture and use. FmocLys(ivDde)-OH is incorporated as residue 75 tofacilitate site-specific labelling at this position of the protein (SEQID NO: 12). The naturally occurring lysine at position 75 is modifiedthrough biotinylation. A suitable spacer, such as a PEG spacer, may beincorporated between the ε-amino functionality and the biotin (SEQ IDNO: 13):

Thus, in certain embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 13:

XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP

X1=pyroGlu (but may remain as Gln in some embodiments)

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 9 below). Themodified CCL11 (Eotaxin) corresponds to residues 1 to 74 of the fulllength mature protein (and lacks the N-terminal signal peptide of 23amino acids, which is cleaved off) and thus retains the chemokine fold(SEQ ID NO: 14). The lysine at position 73 may be modified throughbiotinylation. FmocLys(ivDde)-OH is incorporated as residue 73 tofacilitate site-specific labelling at this position of the protein (SEQID NO: 15). A suitable spacer, such as a PEG spacer, may be incorporatedbetween the ε-amino functionality and the biotin. The biotinylatedversion comprises, consists essentially of or consists of the amino acidsequence of SEQ ID NO:16.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16:

SEQ ID NO: 14 GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

SEQ ID NO: 16 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(PEG-Biotin)

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 10 below). Themodified CCL5 (RANTES) corresponds to residues 1 to 68 of the fulllength mature protein (and lacks the N-terminal signal peptide of 23amino acids, which is cleaved off) and thus retains the chemokine fold.The single methionine (Met67) within the sequence is mutated to lysine,to mitigate against oxidation of this residue during the chain assembly(SEQ ID NO: 17). This Met to Lys substitution provides a lysine atposition 67 which can be modified through biotinylation.FmocLys(ivDde)-OH is incorporated as residue 67 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 18).The biotinylated version comprises, consists essentially of or consistsof the amino acid sequence of SEQ ID NO: 19.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 19:

SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC ANPEKKWVREYINSLEXS

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG (e.g. K(Biotin))

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 11 below). Themodified CCL20 (MIP-3a) corresponds to residues 1 to 70 of the fulllength mature protein (and lacks the N-terminal signal peptide of 26amino acids, which is cleaved off) and thus retains the chemokine fold(SEQ ID NO: 20). FmocLys(ivDde)-OH is incorporated as residue 68 tofacilitate site-specific labelling at this position of the protein (SEQID NO: 21). The naturally occurring lysine at position 68 is modifiedthrough biotinylation. A PEG spacer may be incorporated between theε-amino functionality and the biotin. The final protein may thuscomprise, consist essentially of or consist of the amino acid sequenceof SEQ ID NO: 22.

Thus, in certain embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 22:

ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCAN PKQTWVKYIVRLLSKKVXNM

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, in particularK(PEG-Biotin)

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 13 below). Themodified CXCL11 (ITAC) corresponds to residues 1 to 73 of the fulllength mature protein (and lacks the N-terminal signal peptide of 21amino acids, which is cleaved off) and thus retains the chemokine fold(SEQ ID NO: 26). An additional lysine is inserted at the C-terminus,optionally via a PEG spacer, at position 74. The chemokine may thuscomprise, consist essentially of or consist of the amino acid sequenceof SEQ ID NO: 28.

SEQ ID NO: 28: FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNFX

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG. The amino acid residue maybe added via a spacer molecule such as PEG and may thus be “PEG-K”.

FmocLys(ivDde)-OH is incorporated, followingFmoc-12-amino-4,7,10-trioxadodecanoic acid, as residue 74 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 27).The ε-amino side chain functionality of the additional Lys(74) ismodified through biotinylation. The final protein may thus comprise,consist essentially of or consist of the amino acid sequence of SEQ IDNO: 28 where residue 74 is PEG-K.

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 14 below). Themodified CXCL10 (IP-10) corresponds to residues 1 to 77 of the fulllength mature protein (and lacks the N-terminal signal peptide of 21amino acids, which is cleaved off) and thus retains the chemokine fold.An amino acid which is capable of biotinylation, such as lysine orornithine for example, may be inserted as residue 78. Insertion may bevia a spacer, such as a PEG spacer. The linear amino acid sequence (SEQID NO: 29) is shown, prior to attachment of the PEG spacer, additionallysine and biotin molecules:

VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP

Thus, position 78 may be modified through biotinylation.FmocLys(ivDde)-OH may be incorporated as residue 78 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 30).A suitable spacer, such as a PEG spacer, may be incorporated between theε-amino functionality and the biotin. The biotinylated versioncomprises, consists essentially of or consists of the amino acidsequence of SEQ ID NO: 31.

Thus, in certain embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 31:

VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSPX

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin) and maybe attached via a spacer molecule, e.g. PEG-K(Biotin)

The specific chemokines, including derivatives thereof as describedherein, newly synthesised for use in the methods of the invention mayrepresent separate aspects of the invention. Biotinylation is positionedso as to permit immobilisation whilst retaining receptor bindingcapability.

Chemokines useful in the various embodiments of the invention may besynthesised through any suitable means known in the art. Preferably, thechemokines are chemically synthesised as this facilitates modificationand labelling etc. However, recombinant DNA based approaches may also beemployed in combination with appropriate labelling and modificationtechnologies as required. Thus, in certain embodiments the inventionalso provides a nucleic acid molecule encoding the chemokines of thevarious embodiments of the invention. In certain embodiments theinvention also relates to a vector containing such a nucleic acidmolecule and a host cell containing the vector. The vector mayadditionally comprise a suitable promoter operably linked to the nucleicacid molecule, to facilitate transcription of the corresponding mRNAmolecule. The host cell may be capable of expressing the protein bytranscription and translation of the nucleic acid molecule encoding achemokine of the various embodiments of the invention.

The chemokines useful in the various embodiments of the invention can bebiotinylated by methods known in the art such as described in WO00/50088 A2, which is incorporated herein by reference in its entirety.As indicated above, site-specific labelling of the chemokines of thevarious embodiments of the invention is preferable, although anylabelling technique which does not significantly affect thereceptor-binding capacity of the chemokine may be employed. Varioussite-specifically biotinylated chemokines and native chemokines areavailable commercially, for instance from Almac, Craigavon, UK. Inspecific embodiments the one or more chemokines are biotinylated via aspacer group. The spacer may be employed to prevent the biotin groupfrom impacting on the activity of the chemokine, in particular bindingof the chemokine to its cognate receptor. Any suitable spacer thatfacilitates retention of receptor binding properties of the chemokinemay be employed in the various embodiments of the invention. Thus, inthe specific embodiments described above, spacers other than PEG spacersmay be employed as appropriate. In specific embodiments, the spacer is apolyethylene glycol (PEG) spacer. PEG has been shown to be an effectivespacer permitting attachment of biotin to the chemokine (which can thenbe immobilized on the solid support through interaction withstreptavidin) without compromising receptor binding capability.

In the context of the various embodiments of the present invention theterm “antibody” includes all immunoglobulins or immunoglobulin-likemolecules with specific binding affinity for the relevant chemokinereceptor (including by way of example and without limitation, IgA, IgD,IgE, IgG and IgM, combinations thereof, and similar molecules producedduring an immune response in any vertebrate, for example, in mammalssuch as humans, goats, rabbits and mice). Specific immunoglobulinsuseful in the various embodiments of the invention include IgG isotypes.The antibodies useful in the various embodiments of the invention may bemonoclonal or polyclonal in origin, but are typically monoclonalantibodies. Antibodies may be human antibodies, non-human antibodies, orhumanized versions of non-human antibodies, or chimeric antibodies.Various techniques for antibody humanization are well established andany suitable technique may be employed. The term “antibody” also refersto a polypeptide ligand comprising at least a light chain or heavy chainimmunoglobulin variable region which specifically recognizes and bindsan epitope of an antigen, and it extends to all antibody derivatives andfragments that retain the ability to specifically bind to the relevantchemokine receptor. These derivative and fragments may include Fabfragments, F(ab′)₂ fragments, Fv fragments, single chain antibodies,single domain antibodies, Fc fragments etc. The term antibodyencompasses antibodies comprised of both heavy and light chains, butalso heavy chain (only) antibodies. In specific embodiments, theantibodies may be engineered so as to be specific for more than onechemokine receptor, for example bi-specific to permit binding to twodifferent chemokine receptors. Suitable commercially availableantibodies which bind to the chemokine receptors of interest are listedin table 1 below. They may or may not be labelled. General reference maybe made to “Antibodies a laboratory manual: By E Harlow and D Lane. pp726. Cold Spring Harbor Laboratory. 1988”, which reference isincorporated herein in its entirety.

TABLE 1 Commercially available fluorophore labelled antibodies againstspecific chemokine receptors Antibody Fluorophore Supplier CCR5 PEBiolegend CCR2 PerCP Cy5.5 Biolegend CCR6 PerCP Cy5.5 BD BiosciencesCCR3 PE Biolegend CCR1 Alexa Fluor 647 Biolegend CCR9 APC R&D Systems

Anti-CCR2 antibodies are described for example in WO 2010/021697,incorporated herein by reference. Further examples of potentially usefulantibodies include MLN-1202, an anti-CCR2 monoclonal antibody currentlyundergoing clinical trials (Millennium Pharmaceuticals).

The chemokine receptor expressing cells may thus be targeted usingalternative binding agents, such as antibodies or other chemicalcompounds, as defined herein, rather than the natural chemokine bindingpartner. This approach is a new approach to treating inflammatoryconditions and in particular multiple sclerosis.

Thus, in certain embodiments the invention also provides an apheresiscolumn loaded with a solid support comprising a binding reagent capableof specifically binding to a chemokine receptor immobilized directly orindirectly on the support to permit removal of a cell expressing thechemokine receptor from the peripheral blood of a patient, wherein thebinding reagent is not a chemokine. The binding reagent capable ofspecifically binding to the chemokine receptor may be an agonist or anantagonist of the chemokine receptor. In specific embodiments, thebinding reagent capable of specifically binding to the chemokinereceptor is selected from an antibody and a chemical compound.

In other embodiments the invention thus also provides a method fortreating an inflammatory condition (in particular MS) comprisingapplying peripheral blood from a patient/subject to an apheresis columnas defined above (an apheresis column loaded with a solid supportcomprising a binding reagent capable of specifically binding to achemokine receptor immobilized directly or indirectly on the support topermit removal of a cell expressing the chemokine receptor from theperipheral blood of a patient, wherein the binding reagent is not achemokine) thus removing chemokine receptor expressing cells from theperipheral blood of the patient/subject. The method may comprisereturning the blood depleted of the chemokine receptor expressing cellsto the patient/subject.

Similarly, in other embodiments the invention provides a binding reagentcapable of specifically binding to a chemokine receptor for use in thetreatment of an inflammatory condition, wherein the binding reagent isimmobilized on a solid support contained within an apheresis column asdefined above (an apheresis column loaded with a solid supportcomprising a binding reagent capable of specifically binding to achemokine receptor immobilized directly or indirectly on the support topermit removal of a cell expressing the chemokine receptor from theperipheral blood of a patient/subject, wherein the binding reagent isnot a chemokine), to which is applied peripheral blood from a patientthus removing chemokine receptor expressing cells from the peripheralblood of the patient.

These aspects of the various embodiments of the invention may beintegrated into the more focused therapeutic aspects of the variousembodiments of the invention and thus, the remainder of the disclosure,including all specific embodiments applies mutatis mutandis.

Solid support materials for immobilizing the binding reagents of thevarious embodiments of the invention are known in the art. “Solidsupport” refers to, for example, materials having a rigid or semi-rigidsurface or surfaces, and may take the form of beads, resins, gels,microspheres, or other geometric configurations. A useful supportmaterial is one that does not activate blood cells so as to make themcoagulate or adhere to the support as peripheral whole blood is appliedto the device. In certain embodiments, a support treated with an agentto provide it with anti-coagulation properties, in particular aheparinized support is employed. Alternatively, the blood of the patientmay be treated with an anti-coagulant such as heparin prior toapplication to the support. Useful support materials comprise highmolecular weight carbohydrates, in particular carbohydrates having amolecular weight of 100 kDa or more, such as agarose, in particulateform, optionally cross-linked, and cellulose. Other preferred supportmaterials are polymers, such as carboxylated polystyrene, and glass. Thesupport of the various embodiments of the invention may be provided inthe form of particles or fibres. The support particles may have regularform, such as spheres or beads, or irregular form. They may be porous ornon-porous. A preferred average particle size of the support is from 50μm to 2 mm. In certain embodiments Sepharose™, a cross linked,beaded-form of agarose, is used as column matrix. It is chosen for itsoptimal distribution capacity and can provide a large available area foraffinity binding. Solid supports may be provided in the form of magneticbeads, with the specific binding reagent immobilized on the magneticbeads. Following capture of the (CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9) chemokine receptor expressing cells from the blood, thebeads can be removed from the blood with the aid of an appropriatemagnetic separator.

Methods for immobilizing binding reagents on a solid support are knownin the art. A binding reagent, such as a chemokine, antibody, peptide,nucleic acid or chemical compound, can be immobilized on the support ina direct or indirect manner. Immobilization can be by means of asuitable linker in some embodiments. A preferred method of indirectimmobilization of a binding reagent, such as a chemokine, relies uponthe interaction between biotin and avidin molecules. “Avidin” or “avidinmolecule” refers to any type of protein that specifically binds biotinto the substantial exclusion of other (small) molecules that might bepresent in a biological sample. Examples of avidin include avidins thatare naturally present in egg white, oilseed protein (e.g., soybeanmeal), and grain (e.g., corn/maize), and streptavidin, which is aprotein of bacterial origin. Thus, biotinylation of the binding reagentand use of an avidin molecule such as streptavidin immobilized on thesolid support allows reliable attachment of the binding reagent to thesolid support according to methods known in the art. Specifically, sucha method may comprise providing the binding reagent in biotinylatedform, providing a solid support having streptavidin immobilized on itssurface, contacting the support with an aqueous solution of thebiotinylated binding reagent, and rinsing the support with an aqueoussolvent. In addition, binding pair interactions, such asantibody—antigen interactions, may also be utilised for indirectimmobilisation of binding reagent onto a support. In such embodimentsthe support may be derivatised with one member of a binding pair, suchas an antibody or fragment or derivative thereof, as defined herein,which has known affinity for a particular peptide sequence or smallmolecule hapten. Incorporating the other member of the binding pair,such as an antigen, peptide sequence or the hapten onto or into thebinding reagent facilitates immobilisation onto a solid support coatedwith the corresponding antibody or fragment or derivative thereof. Thus,the binding reagent may be modified to include the peptide sequence orhapten into the linear molecule or may be added as a side chain orlabel. Any suitable antibody-antigen pair may be employed. The antibodyfragment or derivative may be any fragment or derivative that retainsspecific binding affinity for the appropriate antigen. Examples includeFab, F(ab′)₂ fragments, scFV, VH domains, single domain antibodies (suchas nanobodies), heavy chain antibodies and humanized version ofnon-human antibodies etc. Other high affinity interactions can beutilised for immobilisation of binding reagents, as long as the bindingreagent can be synthesised or derivatised with one of the interactingpartners and the solid support synthesised or derivatised with the otherinteracting partner without loss of binding activity (i.e. binding ofthe binding reagent to the appropriate chemokine receptor).Immobilization may occur via essentially the same interaction in reversein some embodiments. Thus, the binding reagent which may be a chemokinefor example, may be attached to an antibody as defined herein, and thesolid support derivatised with the antigen. The chemokine may beproduced as a fusion protein with the antibody.

Alternatively binding reagents, such as chemokines and antibodies, canbe immobilised directly onto a solid support using bioconjugationtechniques established in the field. For example direct immobilisationof proteins onto cyanogen bromide activated solid supports via aminofunctionalities within the primary sequence of the protein.Alternatively, sulphydryl functionalities within proteins can be used todirectly immobilise the protein to alkyl halide derivatised supports orsupports containing free thiol functionalities. In further embodiments,proteins containing α-thioester functionalities can be directlyimmobilised on supports containing 1,2 amino thiol moieties (egN-terminal cysteine) using the native chemical ligation reaction.Alternatively proteins modified with ketones and aldehydes can beimmobilised on solid supports derivatised with hydrazinyl, hydrazide andaminoxy functionalities using hydrazone/oxime bond forming ligationreactions (and vice versa). Alternatively ‘Click’ chemistry can be usedto immobilise proteins onto solid supports, whereby the protein and thesupport are derivatised with the appropriate mutually reactive chemicalfunctionalities (azides and alkynes). In other embodiments Staudingerligation chemistry can be used to immobilise appropriately derivatisedproteins onto the appropriately derivatised solid supports.

The solid support is contained within or carried by the apheresiscolumn. Thus, by “loaded” is meant that the column carries or containsthe solid support in a manner such that (peripheral) blood can flowthrough the column in contact with the solid support. Thus, the solidsupport provides a matrix within the column through which blood flows,in continuous fashion in certain embodiments. This permits cellsexpressing the specific chemokine receptor to be removed from the bloodpassing through the column, such that blood exiting the column isdepleted of the specific chemokine receptor-expressing cells. Inspecific embodiments, the apheresis column is loaded with a supportcomprising streptavidin immobilized on the support and one or morebiotinylated binding reagents, such as chemokines, bound to thestreptavidin on the support. The solid support may be comprised of ahigh-molecular weight carbohydrate, optionally cross-linked, such asagarose.

As discussed above, the binding reagent is coupled to the solid support.The relative amounts of binding reagent may be controlled to ensure thatcoupling between the solid support and the binding reagent will beimmediate, minimising the risk of binding reagent decoupling from thesolid support. Thus, it may be ensured that there is a relative excessof immobilization sites for the binding reagent on the solid support.Alternatively, or additionally, following immobilization of the bindingreagent on the solid support, the solid support may be washed to removeany unbound binding reagent.

The apheresis column utilised in the various embodiments of the presentinvention acts as a leukapheresis treatment for conditions associatedwith multiple sclerosis. The column acts to specifically remove one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9-expressingmonocytes by exploiting the interaction between CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expressed on the cell surface and a specificbinding reagent immobilized on a solid support contained within orcarried by the column. The overall column typically comprises, consistsof, or consists essentially of three combined components; 1) a housingwhich contains or carries 2) the solid support and 3) one or morebinding reagents (immobilized thereon) which specifically bind one ormore chemokine receptors. The housing may be manufactured from anysuitable material for clinical use. In certain embodiments the housingis composed of a plastic material. The housing includes an in flow sitefor entry of blood and an out flow site for blood (depleted of targetcells) to exit the column. The housing may be designed to maintain acontinuous blood flow through the solid support matrix. The housing (asshown for example in FIG. 3) may include a top portion which comprises adistribution plate (2) at the inflow site (1) to spread the blood evenlyover the entire matrix area. The distribution plate may act as a firstsafety barrier preventing larger particles flowing through the columnand into the patient. However, the distribution plate is not essentialand may be removed in some embodiments to decrease the overallresistance in the system. The column may contain one or more safetyfilter units (3 and 4) placed at the inflow (1) and/or outflow (5) sitesof the plastic housing. Such filter units may act to prevent particleslarger than blood cells passing in and/or out of the column. The safetyfilter units may contain a plurality of filters, such as two, three orfour filters designed to be a robust barrier and stop all particleslarger than blood cells passing through the column. Inclusion of safetyfilters (3 and 4) at both ends of the column serves to minimize the riskof leakage of particles into the patient, including in the event thatthe device is incorrectly connected resulting in blood flow in theopposite direction to that intended. The safety filters may comprise ofany suitable pore size to prevent particles larger than blood cells frompassing through the column, as would be readily understood by oneskilled in the art. Suitable filters are commercially available. Inspecific embodiments, the pore size of the filter(s) is betweenapproximately 60 μm and 100 μm, more specifically approximately 80 μm.The solid support and binding reagent components are discussed infurther detail herein.

The volume of the housing may be varied depending upon the blood volumesintended to pass through the column. Typically, the volume of thehousing is between approximately 40 ml and 200 ml, more specifically 50ml to 150 ml or 60 ml to 120 ml.

The column is generally applied in the form of an apheresis circuit. Inthis context, the overall system includes the apheresis column, tubingand an appropriate pump to pump the blood around the circuit. The systemis illustrated in FIG. 4. The patient (1) is connected to theextracorporeal circuit via sterile needles to veins in the right and theleft arms. A saline bag (3) is also connected and the saline solution ispumped with a suitable pump (2). Blood is drawn from one arm of thepatient through the sterile tubing system by the blood pump (4) andpassed through the column (6) and back to the patient. The tubing systemmay be connected to the column via any suitable coupling, such asstandard dialysis luer-lock couplings. The couplings on the column maybe colour-coded for correct assembly. For example, red tubing for inflowto the red column top and blue tubing for outflow back to the patient.An air detector (8) may be present in the circuit. Inlet pressure (5)and/or Pven sensors (7) may additionally be employed to monitor thepressure in the circuit.

An apheresis pump, such as the 4008 ADS pump manufactured by FreseniusMedical Care or the Adamonitor pump, may monitor the patient's inflowand outflow. The pump may also monitor the pressure in theextracorporeal circulation. The pump may be able to discriminate air bya bubble catcher and air detector. A clot catcher filter may bepositioned inside the bubble catcher. The pump may also incorporate anoptical detector to distinguish between light, e.g. saline solution orair present in the tubing system and dark e.g. blood present in thetubing system.

A schematic diagram of a suitable pump, showing the air detector andoptical filter is shown in FIG. 5. If the pump system detects airbubbles and optical fluctuations or if extracorporeal pressure valuesare out of the set range, then the pump may stop immediately.Alternatively or additionally a visual/audible alarm may be emitted.

The treatment methods of the various embodiments of the invention maythus rely upon an extracorporeal circuit. The methods may be consideredas ex vivo or in vitro methods and be defined solely with reference tosteps performed outside of the patient. In some embodiments, however,the method further comprises, prior to application of the blood to thecolumn, collecting peripheral blood from the patient. In a furtherembodiment, the method further comprises, following the application ofthe blood to the column, infusing the blood depleted of (CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9) chemokine receptor expressing cellsto the patient. This is then a complete leukapheresis treatment method.Thus, a leukaphereis method, for treating multiple sclerosis, comprisescollecting peripheral blood from the patient; applying the peripheralblood to an apheresis column loaded with a solid support comprising oneor more binding reagents capable of specifically binding to one or morechemokine receptors, in particular the chemokine receptor CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9, immobilized directly or indirectlyon the support thus removing one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expressing cells from the peripheral blood ofthe patient; and infusing the depleted blood (of chemokine receptorexpressing cells) to the patient.

The peripheral blood may be continuously collected from the patient.Similarly, the depleted blood may be continuously infused to thepatient, through use of an appropriate circuit as described herein.Thus, the support may be disposed in a column through which the blood ismade to flow. This may be achieved using a suitable pump for example, asalso described herein. Blood flow through the column enables the bindingreagent(s) immobilized on the solid support to capture the cellsexpressing the chemokine receptor, thus depleting them from the bloodand preventing their contribution to the (inflammatory) multiplesclerosis.

The methods of the various embodiments of the invention and bindingreagents for use in the methods of the various embodiments of theinvention may require that the patient has been selected for treatmenton the basis of detecting an increase in the level of chemokinereceptor, in particular, one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 expressing cells in a sample obtained from thepatient. Such companion diagnostic methods are described in greaterdetail herein and are based, for example, on the observation that CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expression may be elevated inpatients with multiple sclerosis. More specifically, it is shown hereinthat levels of CCR2 and CCR6 expressing leukocytes, in particular Tlymphocytes, are increased in MS patients (compared with healthycontrols).

Thus, (in this context) in certain embodiments the invention alsoprovides a method of diagnosing, monitoring progression of, ormonitoring treatment of multiple sclerosis comprising determining:

a) the levels of one or more of the chemokine receptor CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cellsb) levels of expression of one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9; and/orc) levels of cells with high expression of one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9in a sample obtained from a subject, wherein high levels of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells,high levels of expression of one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 or high levels of cells with high expression ofone or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 orincreased levels of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 expressing cells compared to control, increased levels ofexpression of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 compared to a control or increased levels of cells with highexpression of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 compared to a control indicate the presence or progression ofmultiple sclerosis. Levels of chemokine receptor expression, as opposedto cell numbers, may also be investigated as increased levels ofchemokine receptor expression per cell may also be diagnosticallyrelevant. The cells may be lymphoctes, in particular T cells.

“Diagnosing” is defined herein to include screening for adisease/condition or pre-indication of a disease/condition, identifyinga disease/condition or pre-indication of a disease/condition andchecking for recurrence of disease/condition following treatment. Themethods of the various embodiments of the invention may also haveprognostic value, and this is included within the definition of the term“diagnosis”. The prognostic value of the methods of the variousembodiments of the invention may be used as a marker of potentialsusceptibility to multiple sclerosis by identifying levels of one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressionlinked to conditions associated with an multiple sclerosis. Thuspatients at risk may be identified before the disease has a chance tomanifest itself in terms of symptoms identifiable in the patient. Incertain embodiments, diagnosis may be made in conjunction with otherobjective indicators of multiple sclerosis. Thus, in specificembodiments, diagnosis is made in conjunction with one or more of thefollowing indicators: clinical measures, multiple sclerosis severityscore, MRI and ologoclonal immunoglobulin pattern in a suitable samplesuch as liquor.

“Monitoring progression of” includes performing the methods to monitorthe stage and/or the state and progression of the multiple sclerosis.Monitoring progression may involve performing the diagnostic methodsmultiple times on the same patient to determine whether the levels ofone or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells are increasing, decreasing or remaining stable over acertain time period. This may be in the context of a treatment regime.

“Monitoring the success of a particular treatment” is defined to includedetermining the levels of one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 expressing cells before and after a treatment. Thetreatment is generally one aimed at treating multiple sclerosis and maybe a treatment according to one of the methods of the variousembodiments of the invention as defined herein. Successful treatment maybe determined with reference to a decrease in one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells as a result of, orfollowing, the treatment. Thus, in such methods a level of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells isdetermined prior to treatment. This level is recorded and a furtherassessment made at a predetermined time following the treatment. Thecomparison of levels of one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 expressing cells permits the success of the treatmentto be monitored. In specific embodiments, a single treatment issufficient to cause a depletion of around 10%, 20%, 30%, 40%, 50%, 60%or 70%, or higher, up to 80%, 90%, 95% or more, or any range of valuesbetween and including these amounts, of one or more specific chemokinereceptors, in particular one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9, expressing cells from the peripheral blood of thepatient. In specific embodiments, at least around 50% depletion isachieved in a single treatment. Thus, successful treatment may bedefined with reference to depletion of one or more of CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cells. Treatment may lead todepletion of between approximately 100 and 500 million one or more ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells, suchas monocytes or lymphocytes, in particular T-cells, in certainembodiments. Additional factors may be included to determine successfultreatment. For example, a lack of increase in one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells following treatmentmay indicate successful treatment in terms of preventing furtherprogression of the condition, optionally combined with an improvement inother markers or staging of the multiple sclerosis.

In specific embodiments, the multiple sclerosis is selected from activeand stable relapsing-remitting multiple sclerosis.

The sample in which one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 expressing cell levels, levels of expression of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 and/or levels ofcells with high expression of one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi), CCR6^(hi), CCR3^(hi),CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi), CCR6^(hi), CCR3^(hi),CCR5^(hi), CCR1^(hi) or CCR9^(hi)) are determined may comprise anysuitable tissue sample or body fluid sample. Generally, the test sampleis obtained from a human subject. Typically, the sample is a bloodsample, in particular a peripheral blood sample. The sample may comprisecerebrospinal fluid (liquor) in certain embodiments. The methods mayinvolve determining levels of one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expressing monocytes, macrophages or lymphocytesin certain embodiments, such as CCR2 and/or CCR6 expressing T-cells.

Levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells, levels of expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 and/or levels of cells with high expression of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi), CCR6^(hi),CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)) may bedetermined according to any suitable method. For example, flow cytometrymay be employed in order to determine the number of cells expressingCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 in the sample, todetermine levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression and/or to identify levels of CCR2^(hi), CCR6^(hi), CCR3^(hi),CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi) cells. Flow cytometrictechniques are described herein and examples of commercially availableantibodies suitably labelled for use in flow cytometry are set out inTable 1 for example. Alternatively, the method may involve steps ofcollecting and fixing the cells in the sample, followed by incubationwith a suitable binding reagent that binds specifically to the CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 chemokine receptor expressingcells in the sample. Any suitable binding reagent, as defined herein,may be employed. For example, a CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 specific antibody may be employed. A wash step may beadopted following an incubation period to remove any unbound reagent.Suitable wash steps and incubation conditions would be well known to oneskilled in the art. The binding reagent may be directly labeled in orderto permit antibody binding to be directly determined. Alternatively asecondary binding reagent, such as an antibody, may be employed whichbinds to the first binding reagent and carries a label. Again, suitableincubation conditions and wash steps would be apparent to one skilled inthe art. The primary and secondary binding reagents may form two halvesof a binding pair. The binding interaction should not prevent theprimary binding reagent binding to the CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 receptor expressing cells, unless a competition assayis being employed. The two halves of a binding pair may comprise anantigen-antibody, antibody-antibody, receptor-ligand,biotin-streptavidin pair etc. in certain embodiments. Other techniquesused to quantify chemokine (CCR2) receptor expressing cell levels, toquantify levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression and/or to quantify levels of CCR2^(hi), CCR6^(hi), CCR3^(hi),CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi) cells includePCR-based techniques such as QT-PCR and protein based methods such aswestern blot. Quantitation may be achieved with reference to fixed celllines carrying known numbers of various receptor expressing cells and/orknown levels of receptor expression per cell. Such fixed cell lines areavailable commercially (for example ChemiScreen™ cell lines fromMillipore). Methods analogous to the treatment methods of the variousembodiments of the invention may also be employed, with binding of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells to the solidsupport being determined following peripheral blood being passed throughthe column.

The levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells, levels of expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 and/or levels of cells with high expression of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi), CCR6^(hi),CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)) may bedetermined relative to a suitable control. When diagnosing multiplesclerosis, a threshold level of cells, level of expression of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 and/or level of cells withhigh expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9(defined as CCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi),CXCR3^(hi) and/or CCR9^(hi)) may be set at or over which a positivediagnosis is made. This threshold may be determined based upon measuringlevels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells, levels of expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 and/or levels of cells with high expression of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi), CCR6^(hi),CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)) in samplesobtained from diseased patients and comparing these levels with levelsof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells,levels of expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9and/or levels of cells with high expression of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi), CCR6^(hi), CCR3^(hi),CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)) in samples obtainedfrom healthy subjects.

In certain embodiments, multiple sclerosis is diagnosed on the basis oflevels of chemokine receptor expressing cells, such as CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cells. A positive diagnosis maybe made in subjects based upon the presence of greater than about 10%,greater than about 20%, greater than about 30%, greater than about 40%,greater than about 50%, greater than about 55%, greater than about 60%,greater than about 65%, greater than about 70%, greater than about 75%,greater than about 80%, greater than about 85%, greater than about 90%,greater than about 95%, or more chemokine receptor expressing cells inthe sample, as a percentage of total cells in the sample. In otherembodiments, multiple sclerosis is diagnosed on the basis of thepresence of a about a 1.2 fold or greater increase, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inchemokine receptor expressing cells, relative to healthy controls.

In specific embodiments, multiple sclerosis is diagnosed on the basis oflevels of CCR2 or CCR6 expressing cells, such as lymphocytes and inparticular T lymphocytes. A positive diagnosis may be made in subjectsbased upon the presence of greater than about 7%, greater than about 10%or greater than about 15% CCR2 expressing T cells in the sample, as apercentage of total cells in the sample. A positive diagnosis may bemade in subjects based upon the presence of greater than about 18%,greater than about 20% or greater than about 22% CCR6 expressing T cellsin the sample, as a percentage of total cells in the sample. Multiplesclerosis may also be diagnosed on the basis of the presence of a abouta 1.2 fold or greater increase, such as about a 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 10, 20, 50 or 100 or greater fold increase in CCR2 or CCR6expressing cells, such as lymphocytes and in particular T lymphocytes,relative to healthy controls.

In certain embodiments, progression of multiple sclerosis, which may bein the context of a treatment regime, is monitored on the basis oflevels of chemokine receptor expressing cells at different time points.Progression of multiple sclerosis may be indicated in subjects basedupon an increase of greater than about 3%, greater than about 4%,greater than about 5%, such as an increase of greater than about 6%,greater than about 7%, greater than about 8%, greater than about 9%,greater than about 10%, greater than about 12%, greater than about 15%,greater than about 20%, greater than about 25%, greater than about 30%,greater than about 35%, greater than about 40%, greater than about 45%or more chemokine receptor expressing cells in the sample, as apercentage of total cells in the sample, compared to a sample taken fromthe same subject at an earlier time point. In other embodiments,progression of multiple sclerosis is confirmed on the basis of thepresence of a about a 1.2 fold or greater increase, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inchemokine receptor expressing cells, relative to a sample taken from thesame subject at an earlier time point.

In specific embodiments, multiple sclerosis is monitored on the basis oflevels of CCR2 or CCR6 expressing cells, such as lymphocytes and inparticular T lymphocytes. Progression of the disease, which may be inthe context of a treatment regime, may be indicated in subjects basedupon the presence of an increase of greater than about 3%, greater thanabout 4%, greater than about 5%, such as an increase of greater thanabout 6%, greater than about 7%, greater than about 8%, greater thanabout 9%, greater than about 10%, greater than about 12%, greater thanabout 15%, greater than about 20%, greater than about 25%, greater thanabout 30%, greater than about 35%, greater than about 40%, greater thanabout 45% or more chemokine receptor expressing cells in the sample, asa percentage of total cells in the sample, compared to a sample takenfrom the same subject at an earlier time point. In other embodiments,progression of multiple sclerosis is confirmed on the basis of thepresence of a about a 1.2 fold or greater increase, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inCCR2 or CCR6 expressing cells, such as lymphocytes and in particular Tlymphocytes, relative to a sample taken from the same subject at anearlier time point.

Regression or successful treatment may be monitored based upon similardecreases over various time points. For example, regression orsuccessful treatment may be indicated in subjects based upon a decreaseof about 3%, such as a decrease of about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 20%,about 25%, about 30%, about 35% or more chemokine receptor expressingcells in the sample, as a percentage of total cells in the sample,compared to a sample taken from the same subject at an earlier timepoint. In other embodiments, regression of multiple sclerosis isconfirmed on the basis of the presence of a about a 1.2 fold or greaterdecrease, such as about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or100 or greater fold decrease in chemokine receptor expressing cells,relative to a sample taken from the same subject at an earlier timepoint.

In specific embodiments, multiple sclerosis is monitored on the basis oflevels of CCR2 or CCR6 expressing cells, such as lymphocytes and inparticular T lymphocytes. Regression or successful treatment of thedisease may be made in subjects based upon a decrease of about 3%, suchas a decrease of about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%,about 35% or more CCR2 or CCR6 expressing cells, such as lymphocytes andin particular T lymphocytes in the sample, as a percentage of totalcells in the sample or by a decrease of about 3%, such as a decrease ofabout 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 12%, about 15%, about 20%, about 25%, about 30%, about 35% or moreCCR2 or CCR6 expressing cells, such as lymphocytes and in particular Tlymphocytes chemokine receptor expressing cells in the sample, as apercentage of total cells in the sample, compared to a sample taken fromthe same subject at an earlier time point. In other embodiments,regression of multiple sclerosis is confirmed on the basis of thepresence of a about a 1.2 fold or greater decrease, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold decrease inCCR2 or CCR6 expressing cells, such as lymphocytes and in particular Tlymphocytes, relative to a sample taken from the same subject at anearlier time point.

Suitable software is freely available (such as the R project forstatistical computing) to perform the necessary statistical analysis ofthe data obtained to calculate a useful threshold. The threshold may beset to maximize sensitivity and/or specificity of the test. Performanceof the test in these respects may be measured by plotting a receiveroperating characteristics (ROC) curve (sensitivity versus specificity).The area under the curve provides an indication of the overallperformance of the test. Thus, once thresholds have been set fordiagnosing the condition, a separate control experiment does notnecessarily have to be run each time a sample is tested. Ratherreference can simply be made to the pre-existing thresholds to determinethe diagnosis. However, in certain embodiments, the sample is testedtogether with a control sample taken from a healthy subject to provide acomparator based upon essentially identical experimental conditions. Thetest sample is generally tested in parallel with the control sample. Thetest sample level of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells, levels of expression of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 and/or levels of cells with high expression of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi),CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi))can then be compared with that of the control sample to make thediagnosis. A control sample from a disease patient may also be tested incertain embodiments. Reference to controls permits relative levels(“high”, “low” etc.) of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells in the test sample to be readily identified and thesignificance thereof interpreted. Reference to controls also permitsrelative levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression (“high”, “low” etc.) within the cell population to bedetermined and the significance thereof interpreted. Such determinationmay, for example, indicate the average levels of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expression per cell in the test sample.

Thus, in specific embodiments, high or higher levels of one or more ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells or highor higher levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression, for example average CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 expression per cell, or high or higher levels of one or moreof CCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi)and/or CCR9^(hi) cells correlate with active disease or more activemultiple sclerosis. Similarly, lower or low levels of one or more ofCCR2, CCR6, CCR3, CCR5, CCR1 and CCR9 expressing cells, or low or lowerlevels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expression,for example average CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression per cell, or low or lower levels of one or more of CCR2^(hi),CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi) and CCR9^(hi) cells maycorrelate with a lack of active inflammation or multiple sclerosis. Thismay be defined as “less active disease”. It can readily be envisagedthat control samples may be assessed and levels of CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cells, levels of expression ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 and/or levels of cellswith high expression of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9(defined as CCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi),CXCR3^(hi) and/or CCR9^(hi)) determined across the range of severitiesof conditions associated with multiple sclerosis. This may assist incorrelating the levels of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 expressing cells, levels of expression of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 and/or levels of cells with high expression ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 (defined as CCR2^(hi),CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi))in the test sample with the relative severity of the condition.

When monitoring progression of, or monitoring treatment of multiplesclerosis, the control samples may be taken from the subject at anearlier time point. They may, however, be based upon known referencevalues as discussed above. Thus, relative levels of CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cells, relative levels of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expression including relativelevels of average CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expression per cell or relative levels of CCR2^(hi), CCR6^(hi),CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi) cells maybe with reference to samples taken from the same subject at a differentpoint in time. In certain embodiments, decreased levels of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cellsdecreased relative levels of one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 expression including decreased relative levelsof average CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressionper cell, or decreased relative levels of one or more of CCR2^(hi),CCR6^(hi), CCR3^(hi) CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/or CCR9^(hi)cells correlate with successful treatment. The treatment may be anysuitable treatment, but in specific embodiments is a treatment accordingto the various embodiments of the invention.

When monitoring progression of multiple sclerosis, increased levels ofone or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells increased relative levels of one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expression including increasedrelative levels of average CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 expression per cell or increased relative levels of one or more ofCCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi) and CCR9^(hi)cells may indicate the progression of condition or disease. Thus, iflevels of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells, levels of expression of one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9 and/or levels of cells with highexpression of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/orCCR9 (defined as CCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi),CXCR3^(hi) and/or CCR9^(hi)) are increased in a sample taken later thana sample from the same patient this may indicate progression of thecondition.

Since the levels of one or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3and/or CCR9 expressing cells, levels of one or more of CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expression or levels of one or more ofCCR2^(hi), CCR6^(hi), CCR3^(hi), CCR5^(hi), CCR1^(hi), CXCR3^(hi) and/orCCR9^(hi) cells are diagnostically relevant, determining such levels ina sample obtained from a subject may influence treatment selection forthat subject. Accordingly, in certain embodiments the invention providesa method of selecting a suitable treatment for multiple sclerosiscomprising determining:

a) the levels of the one or more of chemokine receptor CCR2, CCR6, CCR3,CCR5, CCR1, CXCR3 and/or CCR9 expressing cellsb) levels of expression of one or more of CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9; and/orc) levels of cells with high expression of one or more of CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9in a sample obtained from a subject, wherein high levels of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells,high levels of expression of one or more of CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9 or high levels of cells with high expression ofCCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 or increased levels ofone or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9expressing cells compared to control, increased levels of expression ofone or more of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 comparedto a control or increased levels of cells with high expression of one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 compared to acontrol, result in selection of a treatment as defined herein fortreatment of the multiple sclerosis. In certain embodiments, thechemokine receptor expressing cells are high chemokine receptorexpressing cells, in particular, high CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 expressing cells. The cells may be lymphocytes, inparticular T-lymphocytes. The cells may be CCR2 and/or CCR6 expressingcells, such as T-cells, in specific embodiments.

In specific embodiments, multiple sclerosis is treated on the basis ofmeasuring levels of chemokine receptor expressing cells, such as CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressing cells. Thus, atreatment according to the various embodiments of the invention may beapplied based upon the presence of greater than about 10%, greater thanabout 20%, greater than about 30%, greater than about 40%, greater thanabout 50%, greater than about 55%, greater than about 60%, greater thanabout 65%, greater than about 70%, greater than about 75%, greater thanabout 80%, greater than about 85%, greater than about 90%, greater thanabout 95%, or more chemokine receptor expressing cells in the sample, asa percentage of total cells in the sample. In other embodiments,multiple sclerosis is treated according to the various embodiments ofthe invention on the basis of the presence of a about a 1.5 fold orgreater increase, such as about a 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50or 100 or greater fold increase in chemokine receptor expressing cells,relative to healthy controls.

In specific embodiments, multiple sclerosis is treated on the basis ofmeasuring levels of CCR2 or CCR6 expressing cells, such as lymphocytesand in particular T lymphocytes. Thus, a treatment according to thevarious embodiments of the invention may be applied based upon thepresence of greater than about 7%, greater than about 10% or greaterthan about 15% CCR2 expressing T cells in the sample, as a percentage oftotal cells in the sample. Thus, a treatment according to the variousembodiments of the invention may be applied based upon the presence ofgreater than about 18%, greater than about 20% or greater than about 22%CCR6 expressing T cells in the sample, as a percentage of total cells inthe sample. Alternatively, multiple sclerosis is treated on the basis ofthe presence of a about a 1.5 fold or greater increase, such as about a2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inCCR2 or CCR6 expressing cells, such as lymphocytes and in particular Tlymphocytes, relative to healthy controls.

For the avoidance of doubt, all embodiments described in respect of themethods of the various embodiments of the invention apply to theseaspects mutatis mutandis and are not repeated for reasons ofconciseness. Specifically, multiple sclerosis may be indicated inconjunction with one or more of the following indicators:

clinical measures, multiple sclerosis severity score, MRI andologoclonal immunoglobulin pattern in a suitable sample such as liquor.

The multiple sclerosis may be selected from active and stablerelapsing-remitting multiple sclerosis. In specific embodiments, thesample is a peripheral blood sample.

The methods and medical uses of the various embodiments of the inventionthus can be tailored to the need of individual patients or groups ofpatients on the basis of the various diagnostic methods of the variousembodiments of the invention. By removing from the circulation one ormore of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 expressingcells, such as monocytes, lymphocytes, neutrophils, macrophages,eosinophils and basophils (in particular T-cells, including CCR2 and/orCCR6 expressing T-lymphocytes), upregulated in multiple sclerosis, animportant factor in the inflammatory process of multiple sclerosis canbe controlled. The method of the invention may be effective in treatingor reversing conditions such as active and stable relapsing-remittingmultiple sclerosis.

The various embodiments of the invention will now be described in moredetail by reference to the following non-limiting embodiments andexamples:

DESCRIPTION OF THE FIGURES

FIGS. 1a, 1b & 1 c—the binding of biotinylized MCP-1 by CD4+, CD8+T-cells and CD14+ monocytes respectively, obtained from peripheral bloodof a healthy donor.

FIG. 2a —binding of MCP-1 to monocytes (dashed line) in peripheral bloodtaken from IBD patients. The graph represents a summary of four tests.

FIG. 2b —binding of CCR2-antibody to monocytes (line) in peripheralblood taken from IBD patients. The graph represents a summary of fourtests.

FIG. 3—The plastic house and top showing the distribution plate (2) andsafety filter units (3 and 4).

FIG. 4—The overall leukapheresis system

FIG. 5—The pump with air detector and optical detector (4).

FIG. 6a —Results of in vitro depletion tests performed on the bMCP-1coupled matrix showing ability to eliminate CCR2-expressing cells fromblood from three healthy donors.

FIG. 6b —Results of in vitro depletion tests performed on thebiotinylated RANTES coupled matrix showing ability to eliminatechemokine receptor-expressing cells from peripheral blood of a healthydonor.

FIG. 6c —Results of in vitro depletion tests performed on thebiotinylated MIP-3a coupled matrix showing ability to eliminateCCR6-expressing lymphocytes from blood from three healthy donors.

FIG. 7—Sequence and biotinylation, via a spacer group, of mature proteinMCP-1 derivative containing Gln to pyroGlu modification

FIG. 8—Sequence and biotinylation, via a spacer group, of mature proteinMCP-1 derivative containing Gln to pyroGlu modification and Met toNorleu substitution

FIG. 9—Sequence and biotinylation, via a spacer group, of truncatedMCP-1 derivative containing Met to Norleu substitution

FIG. 10—Alignment of MCP-1 and MCP-5 amino acid sequences

FIG. 11—Sequence and biotinylation, via a spacer group, of (C-terminal)truncated MCP-5 derivative containing lie to Lys modification

FIG. 12—Sequence and biotinylation, of RANTES derivative

FIG. 13—example of gating criteria for CCR2 expressing monocytes

FIG. 14a —Frequency of CCR2 positive T cells. Bars represent mean andSEM of T cells that express CCR2 in 2 patients and 20 healthy controls.

FIG. 14b —Frequency of CCR6 positive T cells. Bars represent mean andSEM of T cells that express CCR6 in 5 patients and 20 healthy controls.The expression of chemokine receptors and specific cell markers wereanalysed with flow cytometry. The T cells were characterized as CD3positive.

FIG. 15a —Binding of the chemokine bMCP-1 to T cells. Bar representsmean frequency and SEM of MCP-1 binding T cells in 5 patients with MS.

FIG. 15b —Binding of the chemokine bMIP3a to T cells. Bar representsfrequency of MIP3a-binding T cells in 1 patient with MS. Blood wasincubated with biotinylated chemokine and analysed with flow cytometry.The T cells were characterized as CD3 positive.

FIG. 16a —Depletion of CCR2 expressing T cells with SepharoseStreptavidin-matrix conjugated with bMCP-1.

FIG. 16b —Depletion of CCR6 expressing T cells with SepharoseStreptavidin-matrix conjugated with bMIP3a. Blood cells from a patientwith MS were incubated with biotinylated chemokine-SepharoseStreptavidin-matrix. Unbound cells were retrieved by washing the matrix.The cells (After Depletion) were then analysed with flow cytometry andcompared with cells that had not been incubated with bchemokine-matrix(Before Depletion).

DESCRIPTION OF PREFERRED EMBODIMENTS

In secondary progressive MS microglia/MØ present at border of plaquesproduce chemokines MCP-1 and CXCL10 responsible for attracting CCR2 andCXCR3 expressing cells including macrophages and astrocytes.

It is shown herein that subjects suffering from MS exhibit increasedfrequency of chemokine receptor expressing cells in the peripheralblood, in particular CCR2 and CCR6 expressing T lymphocytes, compared tohealthy controls. It is also shown herein that the CCR2 cells can beremoved using a suitable binding reagent, in particular MCP-1 (inbiotinylated form) immobilized on a suitable matrix. Similarly, it isshown herein that (the additional) CCR6-expressing cells can be depletedusing a suitable binding reagent, in particular CCL20 (MIP-31), inbiotinylated form, immobilized on a suitable matrix.

The CCL5 levels are significantly elevated in cerebrospinal fluid of MSpatients with relapsing disease demonstrating that circulating CCL5 isinvolved in recruiting CCL5 binding cells to the brain. These findingsare supported by the enrichment of T cells in cerebrospinal fluidexpressing CCR5 and CCR6 suggesting an active accumalation due to achemokine gradient of CCL5. Therefore eliminating cells normallyattracted to the brain by providing an extracorpeal source of CCL5attached to a column will be useful for the treatment of MS.

Examples 1 and 2 Materials and Methods Isolation of Peripheral BloodLeukocytes.

Heparinized peripheral blood from healthy blood donors or inflammatorybowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloridesolution and washed twice in FACS buffer to obtain a suspension of bloodleukocytes.

Chemokines.

The leukocytes were incubated for 30 min in the dark at 4° C. withbiotinylated and Alexa647 Fluor® labeled MCP-1 (in concentrations 10ng/μL and 50 ng/μL). The cells were then washed with FACS-buffer andanalyzed by flow cytometry. All chemokines used in the Examples wereprovided by Almac Sciences Scotland Ltd, Edinburgh, Scotland.

Flow Cytometry Assay.

The flow cytometry assay was performed on a two laser FACS Caliburcytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousandcells were counted and analysed in each sample. For data analyses, CellQuest Pro software from Becton Dickinson was used.

Example 1—Binding of Monocytes to MCP-1

In the experiment with biotinylated MCP-1 it was found that about 90% ofthe monocytes obtained from peripheral blood of healthy donors had boundto the cytokine after 30 min of incubation (FIG. 1a ), whereas CD4+ andCD8+ lymphocytes had not bound (FIGS. 1b and 1c ).

Example 2

Monocytes were investigated for their expression of CCR2 (FIG. 2b ) andtheir ability to bind MCP-1 (FIG. 2a ). CCR2 expression was noted an allmonocytes with the majority of monocytes expressing high levels, usingan anti-CCR2 antibody (FIG. 2b ). The MCP-1 binding to monocytes shownin FIG. 2a corresponds to the CCR2hi expressing population shown in FIG.2b . Thus, MCP-1 binds favourably to CCR2hi expressing cells.

Example 3—Tailored Leukapheresis Column Design and PropertiesIntroduction

Apheresis is an established treatment used for depletion of bloodcomponents, such as antibodies, low-density lipoproteins (LDL) and bloodcells. Leukapheresis is the apheresis treatment used for removal ofwhite blood cells, leukocytes. The patient is connected to anextracorporeal blood circulating system; the blood is drawn from a veinin one arm, passed through a column device and returned into the otherarm of the patient. Side effects of leukapheresis treatments are varyingfrom mild events like headache, dizziness, hypotension, palpitation andflush seen in 0.1 to 5% of treated patients.

The Column

The column is intended to be used as a leukapheresis treatment formultiple sclerosis. It will specifically remove CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9-expressing leukocytes, in particular monocytes,through the use of a binding reagent, more specifically an MCP-1, MCP-2,MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11(I-TAC), CCL25 and RANTES containing resin, exploiting the CCR2, CCR6,CCR3, CCR5, CCR1, CXCR3 and/or CCR9-chemokine interaction. The columnconsists of three combined components, the plastic house, thestreptavidin (SA) Sepharose™ BigBeads matrix and one or more ofbiotinylated MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9),IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES bound to the matrix. Thetreatment is conducted using the same techniques as a standard apheresisprocedure.

The Plastic House (FIG. 3)

The plastic house, designed to keep a continuous blood flow through thematrix, consists of a transparent body and red-coloured top. The top hasa distribution plate (2) at the inflow site (1) to spread the bloodevenly over the entire matrix area. The plate is the first safetybarrier preventing larger particles flowing through the column and intothe patient. Safety filter units (3 and 4) are placed at the inflow (1)and outflow (5) sites of the plastic housing. The safety filter unitcontains three filters designed to be a robust barrier and stop allparticles larger than blood cells passing through the column. Theplastic housing design is shown in FIG. 3. The design with safetyfilters (3 and 4) at both ends of the column device will minimize therisk of leakage of particles into the patient, including in the eventthat the device is placed up side down with the blood flow in theopposite direction to that anticipated.

Streptavidin Sepharose™ BigBeads

The second component in the device is the affinity matrix calledstreptavidin Sepharose™ BigBeads (Sepharose™ GE Healthcare, Sweden).Sepharose™ is a cross linked, beaded-form of agarose, which is apolysaccharide extracted from seaweed. Sepharose™ and agarose arecommonly used as column matrices in biomedical affinity techniques. Itis chosen for its optimal distribution capacity and can provide a largeavailable area for affinity binding.

Binding Reagent

Coupled to the matrix is the third component of the device, the one ormore binding reagents that bind specifically to CCR2, CCR6, CCR3, CCR5,CCR1, CXCR3 and/or CCR9. One or more chemokines such as those selectedfrom the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MCP-5,MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTESmay be employed. These peptides may be synthetic, engineered versions ofthe human chemokine, which are truncated and biotinylated, but retainbinding activity to the CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9receptor. By biotinylating the engineered chemokine, it is able to bindto the streptavidin molecules in the Sepharose™ matrix. Thebiotin-streptavidin binding is known be one of the strongest biologicalinteractions with a Kd in the order of 4×10⁻¹⁴ M. The calculated ratioof streptavidin:biotin binding sites in the column is 10:1. Therefore,the coupling between the matrix and chemokine will be immediate,minimising the risk of chemokine decoupling from the matrix.

The Apheresis System

To conduct the leukapheresis the following components are needed; thecolumn, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

The Circuit

The system is illustrated in FIG. 4. The patient (1) is connected to theextracorporeal circuit via sterile Venflon needles to veins in the rightand the left arms. A saline bag (3) is also connected and the salinesolution is pumped with an ACD pump (2). Blood is drawn from one arm ofthe patient through the sterile tubing system by the blood pump (4) andpassed through the column (6) and back to the patient. The tubing systemis connected to the column via standard dialysis luer-lock couplings.The couplings on the column are colour-coded for correct assembly; redtubing for inflow to the red column top and blue tubing for outflow backto the patient. An air detector (8) is present. Inlet pressure (5) andPven sensors (7) are employed to monitor the pressure in the circuit.

The 4008 ADS Pump

An apheresis pump, from Fresenius Medical Care, monitors the patient'sinflow and outflow, the pressure in the extracorporeal circulation andcan discriminate air by a bubble catcher and air detector. A clotcatcher filter is placed inside the bubble catcher. The pump also has anoptical detector to distinguish between light, e.g. saline solution orair present in the tubing system and dark e.g. blood present in thetubing system.

A schematic diagram of the pump, showing the air detector and opticalfilter is shown in FIG. 5. If the pump system detects air bubbles andoptical fluctuations or if extracorporeal pressure values are out of theset range, then the pump stops immediately and a visual/audible alarmare emitted.

LEGEND FOR FIG. 5

-   -   1. Monitor    -   2. Holder for waste bag    -   3. Modules (left to right—Blood pump, ACD pump, Air detector)    -   4. Reserve places for further modules    -   5. Absorber holder    -   6. Drip detector    -   7. IV pole

Preparation of the Patient

The patient will be administered anticoagulants prior to each treatmentsession. A sterile saline solution with 5000 IE Heparin will be used forpriming the extracorporeal system, thereafter a bolus injection with4000 IE Heparin will be added into the circuit at the start of eachtreatment session.

Leukapheresis Time and Flow Rate

The apheresis system should be operated at a flow rate of 30-60 mL/min.A treatment is finalised after 1800 mL of blood has been circulated.

Storage Conditions

The column devices should be stored between 1 and 25° C. avoidingfreezing and more elevated temperatures. Stability data >3 monthsindicate no difference in functionality over time or by temperature(room temperature and refrigerated). The columns will be kept inrefrigerated conditions until use. Mechanical damage as those resultingfrom violent vibrations and trauma should be avoided. Column storedoutside of these recommendations should not be used.

Transport Conditions

The column devices will be transported under refrigerated condition,avoiding freezing and more elevated temperatures. Mechanical damage suchas those resulting from violent vibrations and trauma should be avoided.

In-Vitro Depletion of Target Cell Populations—MCP-1

To investigate the ability to eliminate CCR2-expressing cells, in vitrotests have been performed on the bMCP-1 coupled matrix. Blood wascollected from blood donors and passed through the column devicecontaining bMCP-1 coupled matrix. Blood samples were taken before andafter column passage and analyzed by flow cytometry (FACS) for thedepletion of CCR2-expressing cells.

The results demonstrate significant depletion of the target populationCCR2-expressing monocytes post matrix perfusion. Depletion tests wereperformed on blood from three healthy donors. The results are shown inFIG. 6 a.

In conclusion, the in-vitro results demonstrate a specific reduction ofup to 80% of the CCR2-expressing cells by the column. Notably,individuals with fewer CCR2 expressing cells initially achieved lowerdepletion. The remaining levels of monocytes were around 20-30% in eachcase, irrespective of the starting point. Non-CCR2-expressing cellsremained unaffected (data not shown).

In-Vitro Depletion of Target Cell Populations—RANTES

To investigate the ability to eliminate CCR1, 3 and 5-expressing cells,in vitro tests have been performed on the biotinylated RANTES coupledmatrix. Blood was collected from blood donors and passed through thecolumn device containing biotinylated RANTES coupled matrix. Bloodsamples were taken before and after column passage and analyzed by flowcytometry (FACS) for the depletion of CCR1, 3 and 5-expressing cells.

The results demonstrate significant depletion of the target populationchemokine receptor-expressing cells post matrix perfusion. Depletiontests were performed on blood from a healthy donor. The results areshown in FIG. 6 b.

The in-vitro results demonstrate a specific reduction of around 20% ofthe chemokine receptor-expressing cells by the column. Non-CCR1, 3 or5-expressing cells remained unaffected (data not shown).

In-Vitro Depletion of Target Cell Populations—MIP-3alpha

To investigate the ability to eliminate CCR6-expressing cells, in vitrotests have been performed on the biotinylated MIP-3a coupled matrix.Blood was collected from blood donors and passed through the columndevice containing biotinylated MIP-3a coupled matrix. Blood samples weretaken before and after column passage and analyzed by flow cytometry(FACS) for the depletion of CCR6-expressing cells.

The results demonstrate significant depletion of the target populationCCR6-expressing lymphocytes post matrix perfusion. Depletion tests wereperformed on blood from three healthy donors. The results are shown inFIG. 6 c.

The in-vitro results demonstrate a specific reduction of up to around15% of the CCR6-expressing cells by the column. Non-CCR6-expressingcells remained unaffected (data not shown).

The RANTES molecule was synthesized by Almac. The amino acid sequence ofthe biotinylated RANTES molecule is set forth as SEQ ID NO: 17:

H2N- SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEKS-CO2H

This molecule has the naturally occurring methionine at position 67replaced with lysine to facilitate biotinylation at position 67.

The side-chain of Lys 67 was directly biotinylated to given the proteinprimary structure shown in FIG. 12. The protein was folded anddisulphide bonds formed between the first and third cysteine in thesequence and between the 2nd and 4th cysteines.

Example 4—MCP1 Derivatives

MCP-1 has been produced with residue 75 as the site of biotinylation onthe chemokine (numbering based upon the mature protein having the aminoacid sequence of SEQ ID NO: 2). Biotinylation permits immobilization ofMCP-1 on a solid support (via a biotin-avidin interaction). The basicamino acid sequence of MCP-1, including a 23 amino acid leader sequenceis set forth as SEQ ID NO: 1,

MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCPKEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

The amino acid sequence of the mature protein is set forth as SEQ ID NO:2,

QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQKWVQDSXDHL DKQTQTPKT

X=Met or Nleu

The inventors have determined that chemokines may display improvedbinding properties where the chemokine is biotinylated via a spacergroup. The spacer may prevent the biotin group from impacting on thebinding affinity of the chemokine.

Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be3,6,—dioxoaminooctanoic acid. The Gln at the N-terminus of the proteinsis subject to pyroGlu formation under physiological conditions. Thus thefirst glutamine (Gln1) of the sequence will be substituted withpyroglutamine. The molecule will be synthesised as a C-terminal amide(via synthesis on an amide linker). The molecule is shown schematicallyin FIG. 7.

A biotinMCP-1 Met to Nleu analogue will also be synthesised. The singlemethionine within the sequence will be altered to Norleucine, tomitigate against oxidation of this residue during the chain assembly andimprove stability of the final product. This molecule is shownschematically in FIG. 8 and in SEQ ID NO: 2.

Once synthesised, the activity of the various biotinMCP-1 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in aequorin functionalcell-based assay on human CCR2 receptor.

Example 5—Synthesis of a CCR2 Antagonist biotinMCP-1 which Binds to theReceptor without Activation

Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995,181, 63) has been shown for an MCP-1 derivative truncated at theN-terminus. In particular, deletion of residues 1-8, results in bindingto CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis ofCCR2 positive cells (inhibition of chemotaxis IC50 20 nM)

The amino acid sequence of the truncated version is set forth as SED IDNO: 3:

VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHLDKQTQTPKT

A derivative of this truncated version will be synthesised comprisingresidues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleusubstitution and derivatised at the ε-amino side chain functionality ofLys75 with PEG-Biotin (TFA salt). This molecule is shown schematicallyin FIG. 9. The PEG spacer will be 3,6,—dioxoaminooctanoic acid.

Once synthesised, the activity of the various biotinMCP-1 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in aequorin functionalcell-based assay on human CCR2 receptor.

Example 6—Demonstrate Removal of CCR2 Expressing Cells Using anAlternative Chemokine Ligand to MCP-1

CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 inaddition to MCP-1. MCP-5 only binds CCR2 and should be selective in itsremoval of CCR2 expressing cells. MCP5 is a mouse chemokine shown tochemotact human CCR2 cells with EC50<3 nM.

The full length amino acid sequence, including the signal peptide, isset forth as SEQ ID NO: 4

MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPREAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

The amino acid sequence of N-terminal processed MCP-5 chemokine is 82amino acids long and is set forth as SEQ ID NO: 5

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFILEP SCLG

An amino acid sequence alignment suggests that MCP-5 has a C-terminalextension when compared to the amino acid sequence of MCP-1. The resultsof this alignment are shown in FIG. 10. On this basis a C-terminaltruncated version of MCP-5 will be synthesised. This truncated versionwill comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 6:

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFIL

In the truncated version, Ile75 to be substituted with Lys, set forth asSEQ ID NO: 7:

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFKL

Following substitution, the substituted version will be biotinylated atposition 75, a lysine or other suitable residue such as ornithine ordiaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid).The protein will be synthesised on an amide linker to yield a C-terminalamide derivative. This molecule is shown schematically in FIG. 11.

Once synthesised, the activity of the various biotinMCP-5 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in aequorin functionalcell-based assay on human CCR2 receptor.

Examples 7 to 14 General Protocols for Chemokine Synthesis Assembly:

Chemical synthesis of chemokines was performed using standard Fmoc solidphase peptides synthesis (SPPS) techniques on an ABI 433 peptidesynthesiser. DIC (0.5M in DMF) and OxymaPure (0.5M in DMF) were used foractivation, acetic anhydride (0.5M in DMF) for capping, and 20%piperidine in DMF for Fmoc deprotection. Rink Amide resin was utilisedfor the generation of C-terminal amide chemokines and Wang resin forC-terminal acid chemokines. After assembly, the resin was washed withDMF and DCM and then dried in vacuo.

Removal of Dde Protection:

The Dde protecting group was removed by treatment of resin with asolution of 2.5% hydrazine in DMF (200 ml) over a 2 hour period. Theresin was then washed with DMF.

Labelling Steps:

1. Couple Fmoc-8-amino-3,6-dioctanoic acid (PEG)

Resin was swollen in DMF and then a solution ofFmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml,0.5M in DMF) and OxymaPure solution (2 ml, 0.5M in DMF) was added. Themixture was sonicated for 3 hours and then washed with DMF.

2. Capping

The resin was capped with acetic anhydride solution (0.5M in DMF, 10 ml)for 5 minutes and then washed with DMF.

3. Fmoc Deprotection

Fmoc deprotection was carried out by treatment with 20% piperidine inDMF solution (2×50 ml) for 15 minutes each. The resin was washed withDMF.

4. Couple Biotin-OSu

A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (3481 μl) in DMF (10ml) was added to the resin and the mixture was sonicated for 3 hours.The resin was washed thoroughly with DMF and DCM then dried in vacuo.

Cleavage:

Dry resin was treated with TFA (10 ml) containing a scavenger cocktailconsisting of TIS (500 μl), thioanisole (500 μl), water (500 μl), DMS(500 μl), EDT (250 μl), NH₄I (500 μg) and phenol (500 μg) and themixture was stirred at room temperature for 5 hours. The solution wasfiltered into cold ether and the resin rinsed with TFA. The precipitatedpeptide was centrifuged, washed with ether, centrifuged and lyophilised.

Purification Protocol:

The crude peptide was purified by reverse phase HPLC (RP-HPLC) using aJupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimisedgradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrilecontaining 0.1% TFA].

Folding Protocol:

Pure peptide (10 mg) was dissolved into 6M GnHCl (16 ml) and thenrapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRISpH 8.5 (84 ml) containing 0.3 mM GSSG and 3 mM GSH. The mixture wasstirred at room temperature for 24 hours and then analysed by RP-HPLC(Jupiter C18, 250×4.6 mm column, 10-60% B over 30 minutes. Purificationby RP-HPLC using an optimised gradient afforded the desired product.

Example 7—biotinMCP-1 (CCL2)

Target Molecule: MCP-1 derivatised at the ε-amino side chainfunctionality of Lys(75) with PEG-Biotin (TFA salt)

Modifications: Human MCP-1 corresponding to residues 1-76, is initiallyexpressed as 99 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The Gln at theN-terminus of the protein is subject to pyroGlu formation underphysiological conditions. Thus Gln1 of the sequence was substituted withpyroglutamine to prevent mixed species of N-terminal Gln and pyroGlubeing generated. This improves the yield of synthesis and ensures ahomogeneous chemokine preparation through column manufacture and use.The naturally occurring lysine at position 75 was modified throughbiotinylation on the resin. A PEG spacer was incorporated between theε-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 8) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 75 (K):

H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT-NH₂

X=pyroGlu

The engineered MCP-1 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN

X1=pyroGlu

X75=K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 9).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 10):

H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT-NH₂

X1=pyroGlu

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

Functional Assay Data:

biotinMCP-1 was tested for agonist activity in an Aequorin assay againsthCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50for recombinant native MCP-1 is 3.1 nM.

Example 8—biotinMCP-2 (CCL8)

Target Molecule: MCP-2 derivatised at the ε-amino side chainfunctionality of Lys(75) with PEG-Biotin (TFA salt)

Modifications: Human MCP-2 corresponding to residues 1-76, is initiallyexpressed as 99 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The Gln at theN-terminus of the protein is subject to pyroGlu formation underphysiological conditions. Thus Gln1 of the sequence was substituted withpyroglutamine to prevent mixed species of N-terminal Gln and pyroGlubeing generated. This improves the yield of synthesis and ensures ahomogeneous chemokine preparation through column manufacture and use.The naturally occurring lysine at position 75 was modified throughbiotinylation on the resin. A PEG spacer was incorporated between theε-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 11) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 75 (K):

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP-NH₂

X=pyroGlu

The engineered MCP-2 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH₂

X1=pyroGlu

X75=K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 12).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 13):

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH₂

X1=pyroGlu

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

Functional Assay Data:

biotinMCP-2 was tested for activity in an Aequorin assay against hCCR2b,(Euroscreen) and was shown to be a partial agonist with an EC50 value of50.9 nM. c.f. EC50 for recombinant native MCP-2 is 23.5 nM (partialagonist).

Example 9—biotinEotaxin (CCL11)

Target Molecule: Eotaxin derivatised at the ε-amino side chainfunctionality of Lys(73) with PEG-Biotin (TFA salt)

Modifications: Human eotaxin corresponding to residues 1-74, isinitially expressed as 97 amino acids comprising the chemokine fold, anda 23 amino acid signal peptide which is cleaved off. The naturallyoccurring lysine at position 73 was modified through biotinylation onthe resin. A PEG spacer was incorporated between the ε-aminofunctionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 14) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 73 (K):

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

The engineered eotaxin sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 73 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 15).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 16):

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) dataof purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3Da.

Functional Assay Data:

biotinEotaxin was tested for activity in an Aequorin assay againsthCCR3, (Euroscreen) and was shown to be an antagonist with an EC50 valueof 211.8 nM. c.f. EC50 for recombinant native eotaxin is 10.7 nM(agonist).

Example 10—biotinRANTES (CCL5)

Target Molecule: RANTES derivatised at the ε-amino side chainfunctionality of Lys(67) with Biotin (TFA salt)

Modifications: Human RANTES corresponding to residues 1-68, is initiallyexpressed as 91 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The single methionine(Met67) within the sequence was mutated to lysine, to mitigate againstoxidation of this residue during the chain assembly, which was observedduring the synthesis of the natural sequence derivative. This Met to Lyssubstitution provided a lysine at position 67 which was modified throughbiotinylation on the resin.

The linear amino acid sequence (SEQ ID NO: 17) is shown, prior toattachment of the biotin molecule at amino acid 67 (K):

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEKS-OH

The engineered RANTES sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEXS-RESIN

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 67 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 18).Subsequent removal of the ivDde protecting group, followed by couplingof the Biotin, was carried out as described in the general protocolsection. Cleavage, purification and folding protocols were carried outas described to furnish the desired active chemokine (SEQ ID NO: 19).

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEXS-OH

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG (e.g. K(Biotin))

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

Functional Assay Data:

BiotinRANTES was tested for agonist activity in an Aequorin assayagainst hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 11—biotinMIP-3α (CCL20)

Target Molecule: MIP-3α derivatised at the ε-amino side chainfunctionality of Lys(68) with PEG-Biotin (TFA salt)

Modifications: Human MIP-3α corresponding to residues 1-70, is initiallyexpressed as 96 amino acids comprising the chemokine fold, and a 26amino acid signal peptide which is cleaved off. The naturally occurringlysine at position 68 was modified through biotinylation on the resin. APEG spacer was incorporated between the ε-amino functionality and thebiotin.

The linear amino acid sequence (SEQ ID NO: 20) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 68 (K):

H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM-OH

The engineered MIP-3α sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVXNM-RESIN

X=K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 68 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 21).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 22).

H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVXNM-OH

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, in particularK(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMip-3α: obtained=8396.4 Da; expected 8397.0 Da.

Functional Assay Data:

BiotinMIP-3α was tested for agonist activity in an Aequorin assayagainst hCCR6, (Euroscreen) and an EC50 value of 1.6 nM was reported.c.f. EC50 for recombinant native MIP-3α is 1.0 nM.

Example 12—biotinTECK (CCL25)

Target Molecule: TECK (Met to Nleu substitution) derivatised at theε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

Modifications: Truncated form of human TECK corresponding to residues1-74 of the mature protein, which encompasses the sequence correspondingto the chemokine fold. The full length mature protein is 127 amino acids(the signal peptide is 23 amino acids in a 150 amino acid immatureprotein). The single methionine within the sequence was altered toNorleucine, to mitigate against oxidation of this residue during thechain assembly, which was observed during the synthesis of the naturalsequence derivative. The Gln at the N-terminus of the proteins issubject to pyroGlu formation under physiological conditions. Thus Gln1of the sequence was substituted with pyroglutamine to prevent mixedspecies of N-terminal Gln and pyroGlu being generated. This improves theyield of synthesis and ensures a homogeneous chemokine preparationthrough column manufacture and use. The naturally occurring lysine atposition 72 was modified through biotinylation on the resin. A PEGspacer was incorporated between the ε-amino functionality and thebiotin.

The linear amino acid sequence (SEQ ID NO: 23) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 72 (K):

H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAXKLLDARNKVF-OH

X1=pyroGlu or Gln

X64=Norleucine

The engineered TECK sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAXKLLDARNXVF-RESIN

X1=pyroGlu or Gln

X64=Norleucine

X72=K(Dde)

NPKSREVQRANIeKLLDARNK(ivDde)VF-RESIN

FmocLys(ivDde)-OH was incorporated as residue 72 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 24).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine.

The final active chemokine thus has the following sequence (SEQ ID NO:25):

H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAXKLLDARNXVF-OH

X1=pyroGlu or Gln

X64=norleucine

X72=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, such as K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinTECK(Met to Nleu substitution): obtained=8958.5Da; expected 8959.6 Da.

Functional Assay Data:

biotinTECK(Met to Nleu substitution) was tested for agonist activity inan Aequorin assay against hCCR9, (Euroscreen) and an EC50 value of 63.6nM was reported. c.f. EC50 for recombinant native TECK is 67.9 nM.

Example 13—biotinITAC (CXCL11)

Target Molecule: ITAC derivatised with Biotin at the ε-amino side chainfunctionality of an additional Lysine inserted at the C-terminus after aPEG spacer (TFA salt)

Modifications: Human ITAC corresponding to residues 1-73, is initiallyexpressed as 94 amino acids comprising the chemokine fold, and a 21amino acid signal peptide which is cleaved off. A PEG spacer and anadditional lysine were inserted at the C-terminus, and modified throughbiotinylation on the resin. The PEG spacer was incorporated at theC-terminus between the protein and the additional lysine.

The linear amino acid sequence (SEQ ID NO: 26) is shown, prior toattachment of the PEG spacer, additional lysine and biotin molecules:

H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNF-OH

The engineered ITAC sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNFX-RESIN

X is PEG-K(ivDde)

Fmoc-12-amino-4,7,10-trioxadodecanoic acid followed by FmocLys(ivDde)-OHwere incorporated at the C-terminus to facilitate site-specificlabelling with biotin at the ε-amino side chain functionality of theadditional Lys (SEQ ID NO: 27). Subsequent removal of the ivDdeprotecting group, followed by coupling of the Biotin, was carried out asdescribed in the general protocol section. Cleavage, purification andfolding protocols were carried out as described to furnish the desiredactive chemokine (SEQ ID NO: 28).

H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNFX-OH

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin) and maybe attached via a spacer molecule, e.g. PEG-K(Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinITAC: obtained=8866.5 Da; expected 8860.6 Da.

Functional Assay Data:

biotinITAC was tested for agonist activity in an Aequorin assay againsthCXCR3, (Euroscreen) and an EC50 value of 15.7 nM was reported. c.f.EC50 for recombinant native ITAC is 0.7 nM.

Example 14—biotinIP-10 (CXCL10)

Target Molecule: IP-10 derivatised with Biotin at the ε-amino side chainfunctionality of an additional Lysine inserted at the C-terminus after aPEG spacer (TFA salt)

Modifications: Human IP-10 corresponding to residues 1-77, is initiallyexpressed as 98 amino acids comprising the chemokine fold, and a 21amino acid signal peptide which is cleaved off. A PEG spacer and anadditional lysine were inserted at the C-terminus, and modified throughbiotinylation on the resin. The PEG spacer was incorporated at theC-terminus between the protein and the additional lysine.

The linear amino acid sequence (SEQ ID NO: 29) is shown, prior toattachment of the PEG spacer, additional lysine and biotin molecules:

H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP-OH

The engineered IP-10 sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSPX-RESIN

X is K(ivDde), optionally attached via a spacer such as PEG, e.g.-PEG-K(ivDde)

Fmoc-8-amino-3,6-dioctanoic acid followed by FmocLys(ivDde)-OH wereincorporated at the C-terminus to facilitate site-specific labellingwith biotin at the ε-amino side chain functionality of the additionalLys (SEQ ID NO: 30). Subsequent removal of the ivDde protecting group,followed by coupling of the Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine.The final active chemokine thus has the following sequence (SEQ ID NO:31):

H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSPX-OH

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin) and maybe attached via a spacer molecule, e.g. PEG-K(Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinIP-10: obtained=9141.0 Da; expected 9141.9 Da.

Functional Assay Data:

BiotinIP-10 was tested for agonist activity in an Aequorin assay againsthCXCR3, (Euroscreen) and an EC50 value of 8.7 nM was reported. c.f. EC50for recombinant native IP-10 is 4.4 nM.

Example 15—MS Diagnosis and Treatment Based Upon CCR2 and CCR6Expressing T-Cells Materials and Methods 1. Flow Cytometric Analysis ofPeripheral Blood

Peripheral blood from patients with Multiple Sclerosis and healthycontrols was collected in heparin tubes. The red blood cells were lysedusing Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4%paraformaldehyde) for four minutes at 37° C. and Lysing buffer (PBS with10 mM Tris and 160 mM NH₄Cl, pH 7.5) for 15 min at 37° C. The cells werewashed in PBS with 2% Bovine Growth Serum, incubated with 10% humanserum for 15 min at room temperature (RT) and stained with antibodies(Table 2) at 4° C. for 30 min. The cells were analysed with flowcytometry on a FACS Canto flow cytometer with the FACSDiva software (BDBiosciences).

TABLE 2 List of antibodies for flow cytometric analysis. AntibodyFluorophore Supplier CD3 V450 BD Biosciences CCR6 PE BD BiosciencesStreptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend

2. Chemokine Binding Test

Peripheral blood from patients and healthy controls was collected inheparin tubes. The red blood cells were lysed using Fix Buffer(Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) forfour minutes at 37° C. and Lysing buffer (PBS with 10 mM Tris and 160 mMNH4Cl, pH 7.5) for 15 min at 37° C. The cells were washed in PBS with 2%Bovine Growth Serum, incubated with 10% human serum 15 min at roomtemperature (RT) and stained with cell specific antibodies together withbiotinylated chemokine (1 μM) or the corresponding chemokine receptorantibody at 4° C. for 30 min (Table 2). The biotinylated chemokine wasdetected via the interaction between biotin and a fluorophore conjugatedStreptavidin. The samples were analysed by flow cytometry on a FACSCanto flow cytometer with the FACSDiva software (BD Biosciences).

3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

Cells were prepared from peripheral blood (section 1). 1 mL SepharoseBigBeads matrix conjugated with 0.4 mg/mL Streptavidin (GE Healthcare)was washed in 50 mL PBS and added to a 5 mL polystyrene tube (BDFalcon™). Biotinylated chemokine (1 μM) was added to the tube andincubated for 20 min at RT to enable immobilization of the chemokine onthe matrix via the biotin-streptavidin interaction. Next, the cells wereadded to the chemokine-matrix and incubated for 20 min at RT. The cellsthat did not bind to the matrix were removed by washing the matrix withPBS in a sterile 40 um nylon filter (BD Falcon™ Cell Strainer). The flowthrough cells were stained with antibodies (Table 2), analysed with flowcytometry and compared with cells from peripheral blood that had notbeen incubated with the chemokine-matrix.

Results and Discussion 1. Flow Cytometric Analysis of Peripheral Blood

White blood cells from patients with Multiple Sclerosis (MS) wereanalysed for the expression of chemokine receptors with flow cytometry.The MS patients exhibited an increased frequency of circulating T cellsthat expressed the chemokine receptor CCR2, 15% compared toapproximately 5% in healthy blood (FIG. 14a ), based upon flow cytometrydata and binding by an anti-CCR2 antibody. Furthermore, the patients hadan increased frequency of T cells that expressed CCR6 (FIG. 14b ).

2. Chemokine Binding Test

CCR2 binds to the chemokine MCP-1 that mediate migration andinfiltration of inflammatory cells to various tissues. The ligand forCCR6 is MIP3a (CCL20) that can mediate migration of T cells into theCNS. Both these receptors are important in the inflammatory process. Inaccordance with the CCR2 and CCR6 expression, the T cells bound thebiotinylated MCP-1 (bMCP-1) (FIG. 15a ) and bMIP3a (FIG. 15b ).

3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

The CCR2 expressing T cells could be efficiently depleted withbMCP-1-conjugated Sepharose Streptavidin Matrix (FIG. 16a ), and theCCR6 expressing T cells could be depleted with bMIP3a-conjugatedSepharose Streptavidin Matrix (FIG. 16b )

We conclude that the frequency of T cells that express CCR2 and CCR6 isenhanced in MS. These T cells can bind the ligands MCP-1 and MIP3a.Furthermore, the majority of the CCR2 and CCR6 expressing T cells can beremoved with Sepharose Streptavidin matrix conjugated with thecorresponding biotinylated chemokine.

The various embodiments of the present invention is not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the various embodiments of the invention in addition tothose described herein will become apparent to those skilled in the artfrom the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims. Moreover, all embodiments described herein are considered to bebroadly applicable and combinable with any and all other consistentembodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for treating multiple sclerosis in a subject in need thereofcomprising applying peripheral blood from the subject to an apheresiscolumn loaded with a solid support comprising one or more bindingreagents capable of specifically binding to a chemokine receptorselected from chemokine receptor CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 andCCR9 immobilized directly or indirectly on the support, whereby one ormore cells expressing chemokine receptors CCR2, CCR6, CCR3, CCR5, CCR1,CXCR3 and/or CCR9 are removed from the peripheral blood of the subjectand the multiple sclerosis is treated.
 2. The method of claim 1, whereinthe multiple sclerosis is selected from active and stablerelapsing-remitting multiple sclerosis, primary progressive relapsingmultiple sclerosis, secondary progressive relapsing multiple sclerosis,and progressive relapsing multiple sclerosis.
 3. The method of claim 1,wherein the binding reagent is an agonist or an antagonist of CCR2,CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9.
 4. The method of claim 1,wherein the binding reagent is an antibody or a chemokine.
 5. The methodof claim 4, wherein the chemokine is selected from MCP-1, MCP-2, MCP-3,MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC),CCL25, and RANTES.
 6. The method of claim 5, wherein the chemokine isMCP-1 or MCP-5.
 7. The method of claim 6, wherein the chemokine receptoris CCR2.
 8. The method of claim 5, wherein the chemokine is MIP-3 alpha.9. The method of claim 8, wherein the chemokine receptor is CCR6. 10.The method of claim 5, wherein the chemokine is RANTES.
 11. The methodof claim 10, wherein the chemokine receptor is selected from CCR3, CCR1,and CCR5.
 12. The method of claim 1, wherein the one or more cells areselected from monocytes, lymphocytes, neutrophils, macrophages,eosinophils, and basophils. 13.-15. (canceled)
 16. The method of claim1, wherein the subject has increased levels of expression of one or moreof CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 as compared to asubject that does not have multiple sclerosis.
 17. The method of claim1, wherein 20-50% of the subject's blood is applied to the column in asingle treatment.
 18. A binding reagent capable of specifically bindingto chemokine receptor CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9.19. (canceled)
 20. The binding reagent of claim 18, which is anantagonist of CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9,respectively.
 21. The binding reagent of claim 18, which is an antibodyor a chemokine.
 22. The binding reagent of claim 21, wherein thechemokine is selected from MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha(MIP-3a), MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25, and RANTES.23. The binding reagent of claim 22, wherein the chemokine is MCP-1 orMCP-5 and the chemokine receptor is CCR2.
 24. (canceled)
 25. The bindingreagent of claim 22, wherein the chemokine is MIP-3a and the chemokinereceptor is CCR6.
 26. (canceled)
 27. The binding reagent of claim 22,wherein the chemokine is RANTES and the chemokine receptor is CCR3,CCR1, or CCR5. 28.-66. (canceled)
 67. A modified CCL8 (MCP-2) chemokinecomprising the amino acid sequence of SEQ ID NO:
 13. 68. The modifiedCCL8 chemokine of claim 67, wherein the residue at position 75 isbiotinylated via a polyethylene glycol (PEG) spacer group in order topermit immobilization of the chemokine on a solid support.
 69. Themodified CCL8 chemokine of claim 68, wherein the PEG spacer is 3,6-dioxoaminooctanoic acid.
 70. A modified CCL8 chemokine comprising the aminoacid sequence of SEQ ID NO:
 11. 71. (canceled)
 72. A modified CCL11chemokine comprising the amino acid sequence of SEQ ID NO:
 14. 73. Themodified CCL11 chemokine of claim 72, wherein the residue at position 73is biotinylated via a polyethylene glycol (PEG) spacer group in order topermit immobilization of the chemokine on a solid support.
 74. Themodified CCL11 chemokine of claim 73, wherein the PEG spacer is3,6-dioxo aminooctanoic acid.
 75. The modified CCL11 chemokine of claim73, comprising the amino acid sequence of SEQ ID NO:
 16. 76. (canceled)77. A modified CCL5 chemokine comprising the amino acid sequence of SEQID NO: 17 or SEQ ID NO:
 19. 78. The modified CCL5 chemokine of claim 77,wherein the residue at position 67 is biotinylated via a polyethyleneglycol (PEG) spacer group in order to permit immobilization of thechemokine on a solid support.
 79. The modified CCL5 chemokine of claim78, which comprises the amino acid sequence of SEQ ID NO:
 19. 80.(canceled)
 81. A modified CCL20 chemokine comprising the amino acidsequence of SEQ ID NO:
 22. 82. The modified CCL20 chemokine of claim 81,wherein the residue at position 68 is biotinylated via a polyethyleneglycol (PEG) spacer group in order to permit immobilization of thechemokine on a solid support.
 83. The modified CCL20 chemokine of claim82, wherein the PEG spacer is 3,6-dioxo aminooctanoic acid.
 84. Themodified CCL20 chemokine of claim 82, comprising the amino acid sequenceof SEQ ID NO:
 20. 85. (canceled)
 86. A modified CXCL10 chemokinecomprising the amino acid sequence of SEQ ID NO:
 31. 87. The modifiedCXCL10 chemokine of claim 86, wherein the residue at position 78 isadded via a polyethylene glycol (PEG) spacer group.
 88. (canceled) 89.The modified CXCL10 chemokine of claim 87, wherein the PEG spacer is3,6-dioxo aminooctanoic acid.
 90. The modified CXCL10 chemokine of claim87, wherein the residue at position 78 is biotinylated in order topermit immobilization of the chemokine on a solid support.
 91. Themodified CXCL10 chemokine of claim 90, wherein the residue at position78 comprises PEG-K-Biotin.
 92. (canceled)
 93. A modified CXCL11chemokine comprising the amino acid sequence of SEQ ID NO:
 28. 94. Themodified CXCL11 chemokine of claim 93, wherein the amino acid atposition 74 is added via a polyethylene glycol (PEG) spacer group. 95.The modified CXCL11 chemokine of claim 94, wherein the amino acid atposition 74 is biotinylated.
 96. The modified CXCL11 chemokine of claim95, wherein the amino acid at position 74 is lysine.
 97. (canceled) 98.A modified CCL2 chemokine comprising the amino acid sequence of SEQ IDNO: 10 or SEQ ID NO:
 8. 99. The modified CCL2 chemokine of claim 98,wherein the residue at position 75 is biotinylated via a polyethyleneglycol (PEG) spacer group in order to permit immobilization of thechemokine on a solid support.
 100. (canceled)
 101. The modified CCL2chemokine of claim 99, wherein the PEG spacer is 3,6-dioxo aminooctanoicacid.
 102. (canceled)
 103. A modified CXCL10 chemokine comprising theamino acid sequence of SEQ ID NO:
 31. 104. The modified CXCL10 chemokineof claim 103, wherein the residue at position 78 is added via apolyethylene glycol (PEG) spacer group.
 105. (canceled)
 106. Themodified CXCL10 chemokine of claim 104, wherein the PEG spacer is3,6-dioxo aminooctanoic acid.
 107. The modified CXCL10 chemokine ofclaim 104, wherein the residue at position 78 is biotinylated in orderto permit immobilization of the chemokine on a solid support.
 108. Themodified CXCL10 chemokine of claim 107, wherein the residue at position78 comprises PEG-K-Biotin.
 109. (canceled)